Open-top microfluidic device with structural anchors

ABSTRACT

A microfluidic device is contemplated comprising an open-top cavity with structural anchors on the vertical wall surfaces that serve to prevent gel shrinkage-induced delamination, a porous membrane (optionally stretchable) positioned in the middle over a microfluidic channel(s). The device is particularly suited to the growth of cells mimicking dermal layers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of, and claims priority to, co-pendingU.S. Pat. Application Serial No. 17/160,617, filed Jan. 28, 2021, whichis a Continuation of Application Serial No.: 15/781,370 filed Jun. 04,2018, now Pat. No.: 10,961,496 issued Mar. 30, 2021, which is a 371 ofInternational PCT/US16/64814 filed Dec. 02, 2016, now expired, whichclaims priority to Provisional Serial No. 62/263,492 filed Dec. 04,2015, now expired, the contents of which are incorporated herein intheir entirety.

FIELD OF THE INVENTION

A microfluidic device is contemplated comprising an open-top cavity withstructural anchors on the vertical wall surfaces that serve to preventgel shrinkage-induced delamination, a porous membrane (optionallystretchable) positioned in the middle over a microfluidic channel(s).

BACKGROUND

In vitro models involving thick gels, i.e. > 0.2 mm in thickness (andmore typically > 0.5 mm in thickness), have a common problem ofshrinkage due to evaporation of water and tissue-induced stresses, whichcause delamination from surfaces containing the gel. This issue preventsrobust implementation of 3D gels with microfluidic systems, which canintroduce physiologically relevant shear forces and concentrationgradients. In addition, such delamination will prevent any attempt toapply mechanical forces, for example stretching or compressing, on thegel itself.

3D gel in vitro models are currently performed in transwells or wellplates, where shrinkage-induced delamination is not important. Prior artsolutions to gel shrinkage include transplanting the 3D gelpost-shrinking, and pre-compressing the gel, both of which areequivalent to pre-shrinking the gel before its use. Past microfluidicsystems that include mechanical stretching focused on stretching a thinmembrane as opposed to uniformly stretching a thick (> 0.5 mm) gel.

SUMMARY OF THE INVENTION

A microfluidic device is contemplated comprising an open-top cavity withstructural anchors on the vertical wall surfaces that serve to preventgel shrinkage-induced delamination, a porous membrane (optionallystretchable) positioned in the middle over a microfluidic channel(s). Bypreventing delamination, this allows for implementation of 3D gels withmicrofluidic systems. The device is particularly suited to the growth ofcells mimicking dermal layers, allowing for the testing of cosmetics andcandidate drugs (including aerosols).

In one embodiment, the present invention contemplates makingmeasurements relating to electrophysiology of cells in an open-top chip.It is not intended that the present invention be limited by the type ofcells; a variety of excitable cells is contemplated. Moreover, suchmeasurements can be done whether the open-top chip comprises a gel ornot. In one embodiment, the present invention contemplates a device,comprising a first structure defining a first chamber, the first chambercomprising an open top surface region; a second chamber, wherein a firstinterface region is formed between the first chamber and the secondchamber ; a membrane disposed at the first interface region, themembrane including a first side facing the first chamber and a secondside facing the second chamber; one or more cells disposed in at leastone of the first chamber and the second chamber; and one or moreelectrodes. In one embodiment, at least one of the first chamber andsecond chamber comprises a fluidic channel. In one embodiment, at leastone of the one or more electrodes is disposed in physical contact withsaid membrane. In one embodiment, said performing anelectrophysiological measurement comprises performing a patch-clampmeasurement. In one embodiment, said device further comprises a coverdisposed on top of at least part of the said open top surface region,and wherein at least one of the one or more electrodes is disposed inphysical contact with the cover. In one embodiment, at least one of theone or more electrodes is present within the first chamber and crossesthe open top surface region. While not intending to be limited to thecell type, in one embodiment, said cells comprise at least one ofneurons and astrocytes. In another embodiment, said cells comprise atleast one of retinal rods and retinal cones. In one embodiment, saidcells comprise at least one of skeletal muscle, smooth muscle andcardiac muscle.

The present invention also contemplates, in one embodiment, a layeredstructure comprising i) fluidic channels covered by ii) a porousmembrane, said membrane comprising iii) a layer of cells and saidmembrane positioned below iv) a gel matrix. In one embodiment, there isa removable cover over the gel matrix (and/or cells). While notintending to be limited to any particular cell type, in one embodiment,the cells are brain microvascular endothelial cells. In one embodimentof the layered structure, it further comprises neurons on, in or underthe gel matrix. In still another embodiment of the layered structure, itfurther comprises astrocytes on, in or under the gel matrix. Cells canbe positioned in various different places (in or on the layeredstructure). In one embodiment, the layer of brain microvascularendothelial cells is positioned on the bottom of the membrane so as tobe in contact with the fluidic channels. It is not intended that thepresent invention be limited to any particular source of cells. In oneembodiment, the brain microvascular endothelial cells are primary cells.

It is not intended that the present invention be limited to embodimentswith only one gel or gel layer. In one embodiment, the layered structurefurther comprises a second gel matrix (e.g. positioned under saidmembrane).

The gel(s) or coatings can be patterned or not patterned. Moreover, whenpatterned, the pattern need not extend to the entire surface. Forexample, in one embodiment, at least a portion of said gel matrix ispatterned.

To make measurements, electrodes can be included in the layeredstructure, i.e. electrodes configured for measuring theelectrophysiology of cells, such as brain microvascular endothelialcells. Other cells can also be tested (e.g. muscle cells).

It is not intended that the present invention be limited by the natureor components of the gel matrix or gel coating. In one embodiment, gelmatrix comprises collagen. A variety of thickness is contemplated. Inone embodiment of the layered structure, said gel matrix is between 0.2and 6 mm in thickness.

In yet another embodiment, the present invention contemplates amicrofluidic device comprising i) a chamber, said chamber comprising alumen and projections in the lumen, said lumen comprising ii) a gelmatrix anchored by said projections, said gel matrix positioned aboveiii) a porous membrane, said membrane in contact with iv) fluidicchannels. In one embodiment, said membrane comprises cells. Theprojections serve as anchors for the gel. The projections, in oneembodiment, project outward from the side walls. The projections, inanother embodiment, project upward. The projects, in another embodiment,project downward. The projections can take a number of forms (e.g. a Tstructure, a Y structure, a structure with straight or curving edges,etc.). In some embodiments, there are two or more projections; in otherembodiments, there are four or more projections to anchor the gelmatrix. In one embodiment, the membrane is above said fluidic channels.In one embodiment the cells comprise a layer of brain microvascularendothelial cells (BMECs). In one embodiment, the BMECs are positionedon the bottom of the membrane so as to be in contact with the fluidicchannels. In one embodiment, it further comprises neurons on, in orunder the gel matrix. In one embodiment, it further comprises a secondgel matrix (e.g. positioned under said membrane). While not limited tothe nature or source of the cells, in one embodiment, the brainmicrovascular endothelial cells are primary cells. In one embodiment, itfurther comprises pericytes on, in or under the gel matrix. In oneembodiment, said gel matrix is under a removable cover. In oneembodiment, said gel matrix is patterned (or at least a portion of it ispatterned). In one embodiment, the device further comprises electrodes,e.g. electrodes are configured for measuring the electrophysiology ofsaid brain microvascular endothelial cells. In one embodiment, said gelmatrix comprises collagen. In one embodiment, said collagen matrix isbetween 0.2 and 6 mm in thickness.

The present invention also contemplates, in one embodiment, a method oftesting, comprising 1) providing a layered structure comprising i0fluidic channels covered by ii) a porous membrane, said membranecomprising iii) a layer of cells in contact with said fluidic channels,said membrane positioned below iv) a gel matrix, said gel matrix under aremovable cover; and 2) measuring the electrophysiology of said cells. Avariety of cell types can be tested. In one embodiment, the cells arebrain microvascular endothelial cells. In another embodiment, the cellsare muscle cells. In one embodiment of this method, the layeredstructure further comprises v) electrodes configured for measuring theelectrophysiology of said brain microvascular endothelial cells. In oneembodiment, said measuring comprises TEER measurements with saidelectrodes. In one embodiment, said TEER measurements indicate tightcell-to-cell junctions between said brain microvascular endothelialcells. In another embodiment, said measuring of step 2) comprises patchclamp measurements, extracellular electrophysiology measurements,imaging using calcium-sensitive dyes or proteins, or imaging usingvoltage-sensitive dyes or proteins. In one embodiment, said brainmicrovascular endothelial cells express the marker Glut 1. In oneembodiment of this method, said layered structure further comprisesneurons on, in or under said gel matrix. In one embodiment, the layeredstructure further comprises a second gel matrix positioned under saidmembrane.

The present invention contemplates a variety of uses for these devicesand methods. In one embodiment, the present invention contemplates amethod of topically testing an agent (whether a drug, food, gas, orother substance) comprising 1) providing a) an agent and b) microfluidicdevice comprising i) a chamber, said chamber comprising a lumen andprojections into the lumen, said lumen comprising ii) a gel matrixanchored by said projections and comprising cell in, on or under saidgel matrix, said gel matrix positioned above iii) a porous membrane andunder iv) a removable cover, said membrane comprising brainmicrovascular endothelial cells in contact with v) fluidic channels; 2)removing said removable cover; and 3) topically contacting said cellsin, on or under said gel matrix with said agent. In one embodiment, saidagent is in an aerosol. In one embodiment, agent is in a liquid, gas,gel, semisolid, solid, or particulate form.

The present invention also contemplates a skin model in the form of amicrofluidic device or layered structure. In one embodiment, the presentinvention contemplates a device or layered structure comprising i)fluidic channels covered by ii) a porous membrane, said membranecomprising iii) a layer of endothelial cells and said membranepositioned below iv) a gel matrix comprising fibroblasts andkeratinocytes. In one embodiment, the gel matrix (and or cells) iscovered by a removable cover. In one embodiment, the fibroblasts arewithin the gel matrix and the keratinocytes are on top of the gelmatrix. In one embodiment, the keratinocytes comprise more than onelayer on top of the gel matrix.

In one embodiment, the present invention contemplates a microfluidicdevice comprising i) a chamber, said chamber comprising a lumen andprojections into the lumen, said lumen comprising ii) a gel matrixanchored by said projections, said gel matrix comprising fibroblasts andkeratinocytes, said gel matrix positioned above iii) a porous membrane,said membrane comprising endothelial cells in contact with iv) fluidicchannels. In one embodiment, the gel matrix (and/or cells) is covered bya removable cover. In one embodiment, the membrane is above said fluidicchannels and wherein the layer of endothelial cells is positioned on thebottom of the membrane so as to be in contact with the fluidic channels.In one embodiment, the fibroblasts are within the gel matrix and thekeratinocytes are on top of the gel matrix.

The present invention contemplates, in one embodiment, a method oftreating endothelial cells, comprising 1) providing a) an angiogenic orarteriogenic growth factor in solution, b) a layered structurecomprising i) fluidic channels covered by ii) a porous membrane, saidmembrane comprising iii) a layer of endothelial cells in contact withsaid fluidic channels, said membrane position below iv) a gel matrixcomprising fibroblasts and keratinocytes; and 2) introducing saidsolution into said fluidic channels comprising said angiogenic orarteriogenic growth factor so as to treat said endothelial cells. In oneembodiment, the gel matrix (and/or cells) is covered by a removablecover. In one embodiment, prior to said introducing of step 2), thecover is removed.

The present invention also contemplates, in one embodiment, a method oftesting a drug or other agent on keratinocytes, comprising 1) providinga) a candidate drug and b) microfluidic device comprising i) a chamber,said chamber comprising a lumen and projections into the lumen, saidlumen comprising ii) a gel matrix anchored by said projections, said gelmatrix comprising fibroblasts and keratinocytes, said gel matrixpositioned above iii) a porous membrane, said membrane comprisingendothelial cells in contact with iv) fluidic channels; and 2)contacting said keratinocytes with said candidate drug. In oneembodiment, the gel matrix (and or cells) is covered by a removablecover. In one embodiment, prior to said contacting of step 2), saidcover is removed. In one embodiment, the agent or drug is in the form ofan aerosol.

The present invention also contemplates, in one embodiment, microfluidicdevice for simulating a function of a tissue, comprising: a firststructure defining a first chamber, the first chamber comprising a geldisposed therein and including an opened region, said gel comprisingintestinal epithelial cells in or on said gel; a second structuredefining a second chamber; and a membrane located at an interface regionbetween the first chamber and the second chamber, the membrane includinga first side facing toward the first chamber and a second side facingtoward the second chamber, said second side comprising livingendothelial cells. In one embodiment, the gel has a patterned surface.

The present invention also contemplates, in one embodiment, amicrofluidic device for simulating a function of a tissue, comprising: afirst structure defining a first chamber, the first chamber comprising agel disposed therein and including an opened region, said gel comprisingmuscle cells in or on said gel; a second structure defining a secondchamber; and a membrane located at an interface region between the firstchamber and the second chamber, the membrane including a first sidefacing toward the first chamber and a second side facing toward thesecond chamber. In one embodiment, the gel has a patterned surface.

In one embodiment, the present invention contemplates a devicecomprising i) a chamber, said chamber comprising a non-linear lumen,said lumen comprising ii) a gel matrix, said gel matrix positioned aboveiii) a porous membrane, said membrane positioned above one or more iv)fluidic channels. In one embodiment, the fibroblasts are within the gelmatrix and keratinocytes are on top of the gel matrix. In oneembodiment, the keratinocytes comprise more than one layer on top of thegel matrix. In one embodiment, the layer of endothelial cells ispositioned on the bottom of the membrane so as to be in contact with thefluidic channels. In one embodiment, the endothelial cells are primarycells. In one embodiment, the primary cells are small vessel humandermal microvascular endothelial cells. In one embodiment, the primarycells are human umbilical vein endothelial cells. In one embodiment, theprimary cells are bone marrow-derived endothelial progenitor cells. Inone embodiment, the keratinocytes are epidermal keratinocytes. In oneembodiment, the non-linear lumen is circular. In one embodiment, thedevice further comprises a removable cover. In one embodiment, thedevice is a microfluidic device and said fluidic channels aremicrofluidic channels.

In one embodiment, the present invention contemplates a microfluidicdevice comprising i) a chamber, said chamber comprising a circularlumen, said lumen comprising ii) a gel matrix comprising fibroblasts andkeratinocytes, said gel matrix positioned above iii) a porous membrane,said membrane comprising endothelial cells in contact with iv)microfluidic channels. In one embodiment, the membrane is above saidfluidic channels and wherein the layer of endothelial cells ispositioned on the bottom of the membrane so as to be in contact with thefluidic channels. In one embodiment, the fibroblasts are within the gelmatrix and the keratinocytes are on top of the gel matrix. In oneembodiment, the keratinocytes comprise more than one layer on top of thegel matrix. In one embodiment, the endothelial cells are primary cells.In one embodiment, the primary cells are small vessel human dermalmicrovascular endothelial cells. In one embodiment, the primary cellsare human umbilical vein endothelial cells. In one embodiment, theprimary cells are bone marrow-derived endothelial progenitor cells. Inone embodiment, the keratinocytes are epidermal keratinocytes. In oneembodiment, the keratinocytes are human foreskin keratinocytes. In oneembodiment, the matrix comprises collagen. In one embodiment, thecollagen matrix is between 0.2 and 6 mm in thickness.

In one embodiment, the present invention contemplates a method oftreating endothelial cells, comprising 1) providing a) an angiogenic orarteriogenic growth factor in solution, b) a layered structurecomprising i) fluidic channels covered by ii) a porous membrane, saidmembrane comprising iii) a layer of endothelial cells in contact withsaid fluidic channels, said membrane position below iv) a gel matrixcomprising fibroblasts and keratinocytes; and 2) introducing saidsolution into said fluidic channels comprising said angiogenic orarteriogenic growth factor so as to treat said endothelial cells. In oneembodiment, the gel matrix comprises collagen. In one embodiment, thecollagen matrix is between 0.2 and 6 mm in thickness.

In one embodiment, the present invention contemplates a fluidic covercomprising a fluidic channel, said fluidic cover configured to engage amicrofluidic device. In one embodiment, the microfluidic devicecomprises an open chamber, and wherein said fluidic cover configured tocover and close said open chamber. In one embodiment, the fluidic coverfurther comprises one or more electrodes.

In one embodiment, the present invention contemplates an assemblycomprising a fluidic cover comprising a fluidic channel, said fluidiccover detachably engaged with a microfluidic device. In one embodiment,the microfluidic device comprises an open chamber, and wherein saidfluidic cover configured to cover and close said open chamber. In oneembodiment, the open chamber comprises a non-linear lumen. In oneembodiment, the non-linear lumen is circular. In one embodiment, thefluidic cover further comprises one or more electrodes.

In one embodiment, the present invention contemplates a method of makingan assembly, comprising: a) providing a fluidic cover comprising afluidic channel, said fluidic cover configured to engage b) amicrofluidic device, said microfluidic device comprises an open chamber,and wherein said fluidic cover configured to cover and close said openchamber; and b) detachably engaging said microfluidic device with saidfluidic cover so as to make an assembly. In one embodiment, the openchamber comprises a non-linear lumen. In one embodiment, the non-linearlumen is circular. In one embodiment, the fluidic cover furthercomprises one or more electrodes.

In one embodiment, the present invention contemplates a microfluidicdevice comprising i) a chamber, said chamber comprising a lumen, saidlumen comprising ii) a gel matrix comprising at least one of neurons andastrocytes, said gel matrix positioned above iii) a porous membrane,said membrane comprising brain microvascular endothelial cells incontact with iv) microfluidic channels. In one embodiment, the neuronsare on, in or under the gel matrix. In one embodiment, the astrocytesare on, in or under the gel matrix.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an exemplary microfluidic device with a membraneregion having cells thereon according to embodiments of the presentdisclosure.

FIG. 2 is a cross-section of the microfluidic device taken along line102-102 of FIG. 1 , illustrating the membrane separating the firstmicrochannel and the second microchannel.

FIG. 3 illustrates an exploded perspective view of an exemplarycross-section through an open-top microfluidic device according toembodiments of the present disclosure.

FIG. 4 illustrates an exploded perspective view of an exemplarycross-section through an open-top microfluidic device with a removablecover according to embodiments of the present disclosure.

FIG. 5A illustrates a perspective view of an exemplary cross-sectionthrough an open-top microfluidic device according to embodiments of thepresent disclosure.

FIG. 5B illustrates a perspective view of the exemplary open-topmicrofluidic device of FIG. 5A including a gel layer above a membranelayer in an opened region of a top structure according to embodiments ofthe present disclosure.

FIG. 5C illustrates a perspective view of the exemplary open-topmicrofluidic device of

FIG. 5B including placement of a plunger stamp into the opened region ofthe top structure according to embodiments of the present disclosure.

FIG. 5D illustrates a perspective view of the exemplary open-topmicrofluidic device of FIG. 5C including a patterned gel in the openedregion of the top structure and a removable cover disposed above the topstructure according to embodiments of the present disclosure.

FIG. 5E illustrates a perspective view of the exemplary open-topmicrofluidic device of FIG. 5D in an exemplary clamping device accordingto embodiments of the present disclosure.

FIG. 5F illustrates a perspective view of an alternative exemplarycross-section through an open-top microfluidic device according toembodiments of the present disclosure.

FIG. 6 illustrates an exemplary plunger stamp with a patterned surfaceaccording to embodiments of the present disclosure.

FIG. 7 illustrates an exemplary pattern for a plunger stamp according toembodiments of the present disclosure.

FIG. 8A illustrates a top view of an exemplary stretchable open-topmicrofluidic device according to embodiments of the present disclosure.

FIG. 8B illustrates a perspective view of the chip top of the exemplarystretchable open-top microfluidic device of FIG. 8A.

FIG. 8C illustrates a perspective view of the chip bottom of theexemplary stretchable open-top microfluidic device of FIG. 8A.

FIGS. 9 and 10 illustrate exemplary perspective views of cross-sectionsthrough the stretchable open-top microfluidic device of FIG. 8A.

FIG. 11 illustrates a partial top view of an exemplary configuration ofmultiple parallel channels for a stretchable open-top micro-fluidicdevice.

FIG. 12 illustrates a partial top view of an exemplary configuration ofspiral channel for a stretchable open-top microfluidic device.

FIG. 13 shows one plan view embodiment of a device comprising more thanone open-top cavity or chamber, which allows for direct gel dispensing,cell seeding, and treatments (including treatment with aerosols andtopicals).

FIGS. 14A - B shows one embodiment of structural anchors along thecavity/chamber walls in order to prevent shrinkage-induced delaminationof a gel (not shown).

FIG. 15 shows one embodiment of bottom layer microfluidics, which allowfor shear forces, concentration gradients, and vascularization (e.g. ofendothelial cells).

FIG. 16 shows one embodiment of vacuum channels designed to allow foruniform physiological stretching of a thick gel.

FIG. 17 shows one embodiment of an assembled chip, showing the open-topchambers above the fluidics.

FIG. 18 shows the embodiment of FIG. 17 , wherein the membrane ishighlighted in order to illustrate the relationship of the assembledcomponents.

FIG. 19 shows a vacuum channel cross- section design that allows bendingof the wall about the corner.

It is not intended that the figures be limiting. The open-topcavity/chamber can have various geometries other than the one depictedabove: e.g. oval, rectangular slot, ellipse. Structural anchors can havevarious geometries other than the one depicted above. For example, theycan have different ‘head’ geometries and sizes.

Alternatively, the gel can be maintained with a mesh wall ormicro-pillar array. FIGS. 20A - B shows a top view and elevated sideview of one embodiment of a micro-pillar array. FIGS. 21A - B shows atop view and elevated side view of one embodiment of a mesh wall orinsert.

The bottom-layer microfluidics can have various channel geometries otherthan the one depicted above, i.e. the channel height, channel width, andchannel path geometry can be changed. FIG. 22 shows a different designfor the microfluidic channels.

FIGS. 23-26 show various embodiments for microfluidic devices ascontemplated herein that are configured for electrophysiologicalmeasurements (e.g., for example, patch clamp measurements usingtransepithelial electric resistance (TEER)..

FIG. 27 illustrates one embodiment of a top view of an assembledopen-top chip microfluidic device of the device depicted in FIG. 13 .

FIG. 28 illustrates one embodiment of an array of open chambers in anopen-top chip device as contemplated herein.

FIGS. 29A - B illustrates one embodiment of a stretchable open top chipdevice.

FIG. 29A: A bottom structure with a spiral microchannel with an inletwell and and outlet well.

FIG. 29B: A top view of a spiral microchannel configured with a circularvacuum chamber.

FIG. 30 illustrates an exploded view of one embodiment of a stretchableopen top chip device demonstrating the layering of a fluidic top, topstructure and bottom structure.

FIGS. 31A - B illustrates a cut-away view of one embodiment of astretchable open top chip device showing the regional placement of assaycells (e.g., epithelial cells, dermal cells and/or vascular cells).

FIG. 32 illustrates a fully assembled view of one embodiment of astretchable open top chip device.

FIGS. 33A and 33B illustrate exploded views of two embodiments of astretchable open top chip device.

FIGS. 34A and 34B illustrate assembled views of a stretchable open topchip device as depicted in FIGS. 33A and 33B.

FIGS. 35A and 35B respectively illustrate an assembled isometric viewand an exploded view of a tall channel stretchable open top chip device.

FIG. 36 presents a top assembled view of one embodiment of a stretchableopen-top microfluidic chip comprising a fluidic cover and a singlechannel.

FIGS. 37A - B presents a crossectional view of a first embodiment of astretchable open top microfluidic chip along plane A of FIG. 36 .

FIG. 37A: Illustrates a fluidic cover in a closed position.

FIG. 37B: Illustrates a fluidic cover in an open position.

FIGS. 38A - B presents a crossection view of a second embodiment of astretchable open top microfludic chip along plane A of FIG. 36 .

FIG. 38A: Illustrates a fluidic cover in a closed position.

FIG. 38B: Illustrates a fluidic cover in an open position.

FIG. 39 presents an exploded view of the array device depicted in FIG.28 .

DESCRIPTION OF THE INVENTION

A microfluidic device is contemplated comprising an open-top cavity withstructural anchors on the vertical wall surfaces that serve to preventgel shrinkage-induced delamination, a porous membrane (optionallystretchable) positioned in the middle over a microfluidic channel(s).The device can be used in many ways with many types of tissues andcells. For example, the organ mimic device described herein can be usedfor the identification of markers of disease; assessing efficacy ofanti-cancer therapeutics; testing gene therapy vectors; drugdevelopment; screening; and for studies of particular cells (andarrangements of cells). In one embodiment, the device serves as a skinmodel. In this embodiment, the open-top device provides an uncoveredchamber comprising a skin-like, human or animal tissue that can betested with drugs, including topicals and aerosols.

A. Gel-Containing Skin Model

In one embodiment, the present invention contemplates a constructcomprising a “dermis” with fibroblasts embedded in a matrix having athickness between 0.2 and 6.0 mm, e.g. a collagen I gel matrix, and an“epidermis”, which is comprised of keratinocytes, e.g. stratified,differentiated keratinocytes. A matrix such as a collagen gel providesscaffolding, nutrient delivery, and potential for cell-to-cellinteraction. In one embodiment, the construct further comprises afunctional basement membrane, which separates the epidermis from thedermis.

In one embodiment, the present invention contemplates a layeredstructure comprising i) fluidic channels covered by ii) a porousmembrane, said membrane comprising iii) a layer of endothelial cells andsaid membrane position below iv) a gel matrix comprising fibroblasts andkeratinocytes. In a preferred embodiment, the fibroblasts are within thegel matrix and the keratinocytes are on top of the gel matrix. In apreferred embodiment, the keratinocytes comprise more than one layer ontop of the gel matrix. In a preferred embodiment, the layer ofendothelial cells is positioned on the bottom of the membrane and is incontact with the fluidic channels. In a preferred embodiment, thefluidic channels provide shear to said endothelial cells.

It is not intended that the present invention be limited to thethickness of the gel matrix. However, a preferred range of thickness isbetween 0.2 and 6 mm, and more preferably between 0.5 mm and 3.5 mm, andstill more preferably approximately 1-2 mm. In a preferred embodiment,the gel matrix is stretchable. In a preferred embodiment, the gel matrixis stretched in a manner such that the entire gel matrix expands, notjust a portion of the gel matrix (such as only the bottom or top of thematrix). In a preferred embodiment, the gel matrix is stretched byvacuum channels that are designed to provide pneumatic stretching thatis uniform across the thickness of the gel.

In a preferred embodiment, the layered structure is positioned in anopen-top microfluidic device (i.e. a device lacking a top covering),wherein the gel matrix is secured in a chamber of the device by anchors.In a preferred embodiment, the surfaces of the device that contact thegel matrix have been treated to enhance attachment of the gel matrix. Ina preferred embodiment, the surfaces have been plasma treated, i.e. thesurface is activated with ionized gas. It has been found that thesurface treatment, in combination with the anchors, prevent delaminationof the gel from the walls of the chamber.

In one embodiment, the fluidic channels bring one or more compounds thatwill induce the endothelial cells to differentiate. In one embodiment,the fluidic channels comprise a solution comprising a vascularendothelial growth factor (VEGF).

The open-top device provides an uncovered chamber comprising a skin-liketissue that can be tested with topicals and aerosols. In one embodiment,drugs are applied topically or transdermally to the keratinocytelayer(s). As used herein, the term “topical” refers to administration ofan agent or agents (e.g. cosmetic, medication, vitamin, etc.) on theskin. “Transdermal” refers to the delivery of an agent (e.g. cosmetic,medication, vitamin, etc.) through the skin (e.g. so that at least someportion of the population of molecules reaches underlying layers of theskin).

In one embodiment, a candidate cosmetic is applied to the keratinocytelayer(s). As used herein, a “cosmetic” refers to a substance that aidsin the enhancement or protection of the appearance (e.g. color, texture,look, feel, etc.) or odor of a subject’s skin. A cosmetic may or may notchange the underlying structure of the skin.

In this skin model, a layer of endothelial cells (ECs) is positioned onthe underside of the membrane facing the fluidic channels. Endothelialcells and endothelial stem cells will, under appropriate conditions,migrate and differentiate. In terms of migration, while not limited toany particular mechanism, it is believed that this motile process isdirectionally regulated by chemotactic, haptotactic, and mechanotacticstimuli and (where applicable) may require degradation of theextracellular matrix to enable progression of the migrating cells. It isbelieved to involve the activation of several signaling pathways thatconverge on cytoskeletal remodeling. Generally, it is been observed thatthe endothelial cells extend, contract, and progress forward. In apreferred embodiment, ECs are grown on a membrane with a porositysufficient to allow for this cell migration, i.e. through the membrane.

In some embodiments, growth factors or compounds that enhance theproduction of the desired cell type(s) can be added to the perfusionfluid in the fluidic channels. By way of nonlimiting example,erythropoietin stimulates the production of red blood cells, VEGFstimulates angiogenesis, and thrombopoietin stimulates the production ofmegakaryocytes and platelets. “Vascular growth” is defined here as atleast one of vasculogenesis and angiogenesis and includes formation ofone or more of the following: capillaries, arteries, veins or lymphaticvessels. Blood vessel formation de novo (vasculogenesis) and fromexisting vessels (angiogenesis) results in blood vessels lined byendothelial cells (ECs).

Vascular endothelial growth factor (VEGF) is an interesting inducer ofangiogenesis and lymphangiogenesis because it is highly specificendothelial cells. The VEGF family currently comprises seven members:VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF. All membershave a common VEGF homology domain. Signal transduction involves bindingto tyrosine kinase receptors and results in endothelial cellproliferation, migration, and new vessel formation. In a preferredembodiment, VEGF (and/or other known angiogenic or arteriogenic growthfactors) is used to induce EC differentiation, proliferation,infiltration, angiogenesis, vascularization, etc., or any combinationthereof.

It is not intended that the present invention be limited to only onesource or type of endothelial cell (EC). In one embodiment, primary ECsare used in the open-top device. In one embodiment, freshly isolatedsmall vessel human dermal microvascular endothelial cells are employedon the open-top-chip. In one embodiment, an endothelial cell line isemployed. In yet another embodiment, human umbilical vein endothelialcells (HUVECs) are used. In still another embodiment, bonemarrow-derived endothelial progenitor cells are seeded in the chip. Instill another embodiment, stem cells that can differentiate into ECs areused.

It is not intended that the present invention be limited to only oneplace for seeding the open-top-chip with ECs. While placement of ECs onthe underside of the membrane (in contact with the fluidics) ispreferred, placement on the topside of the membrane and placement withinthe gel matrix itself are alternative embodiments. With regard to thelatter, in one embodiment, microfluidic pathways in the gel itself arecreated that are thereafter seeded with the endothelial cells.

For example, in one embodiment, microfluidic vessel networks areengineered by seeding human endothelial cells [e.g. umbilical veinendothelial cells (HUVECs)] into microfluidic circuits formed via softlithography in a type I collagen gel. Native, type I collagen at 6-10mg/mL is of an appropriate stiffness to allow high reproducibility ofvessel microstructure and also enables remodeling through degradationand deposition of extracellular matrix. The lithographic process enablesthe formation of endothelium along the microfluidic channels and theincorporation of living cells within the bulk collagen gel matrix withinthe open-top-chip.

In one embodiment, endothelial cells are seeded into the gel containing(or onto confluent lawns of) human fibroblasts and cultured in thepresence of high levels of ascorbate 2-phosphate to create a tissue-likestructure in which endothelial cells organize into tube-like structures.

It is not intended that the skin model be limited to just one type ofkeratinocyte. Indeed, the model can be used with many types of cells ofthe integumentary system including but not limited to Keratinizingepithelial cells, Epidermal keratinocyte (differentiating epidermalcell), Epidermal basal cell (stem cell), Keratinocyte of fingernails andtoenails, Nail bed basal cell (stem cell), Medullary hair shaft cell,Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair rootsheath cell, Hair root sheath cell of Huxley’s layer, Hair root sheathcell of Henle’s layer, External hair root sheath cell, and Hair matrixcells (stem cell). In one embodiment, human foreskin keratinocytes areemployed.

B. Other Cells and Tissues

A variety of different cells and tissue types can be modeled and testedwith the open-top spacer chip described herein. Indeed, the system canvirtually be adapted to all epithelial tissues. In addition to skin,preferred models include (but are not limited) to Lung, the SmallAirway, the gut, muscle (including skeletal, cardiac and or smoothmuscle, and the Blood Brain Barrier (BBB). Both human and animal cellsare contemplated. Cell types which can be used in the open-top devicesinclude, but are not limited to Wet stratified barrier epithelial cells,such as Surface epithelial cell of stratified squamous epithelium ofcornea, tongue, oral cavity, esophagus, anal canal, distal urethra andvagina, basal cell (stem cell) of epithelia of cornea, tongue, oralcavity, esophagus, anal canal, distal urethra and vagina, Urinaryepithelium cell (lining urinary bladder and urinary ducts); Exocrinesecretory epithelial cells, such as Salivary gland mucous cell(polysaccharide-rich secretion), Salivary gland serous cell(glycoprotein enzyme-rich secretion), Von Ebner’s gland cell in tongue(washes taste buds), Mammary gland cell (milk secretion), Lacrimal glandcell (tear secretion), Ceruminous gland cell in ear (wax secretion),Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweatgland clear cell (small molecule secretion), Apocrine sweat gland cell(odoriferous secretion, sex-hormone sensitive), Gland of Moll cell ineyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebumsecretion), Bowman’s gland cell in nose (washes olfactory epithelium),Brunner’s gland cell in duodenum (enzymes and alkaline mucus), Seminalvesicle cell (secretes seminal fluid components, including fructose forswimming sperm), Prostate gland cell (secretes seminal fluidcomponents), Bulbourethral gland cell (mucus secretion), Bartholin’sgland cell (vaginal lubricant secretion), Gland of Littre cell (mucussecretion), Uterus endometrium cell (carbohydrate secretion), Isolatedgoblet cell of respiratory and digestive tracts (mucus secretion),Stomach lining mucous cell (mucus secretion), Gastric gland zymogeniccell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloricacid secretion), Pancreatic acinar cell (bicarbonate and digestiveenzyme secretion), pancreatic endocrine cells, Paneth cell of smallintestine (lysozyme secretion), intestinal epithelial cells, Types I andII pneumocytes of lung (surfactant secretion), and/or Clara cell oflung.

One can also coat the membrane with Hormone secreting cells, such asendocrine cells of the islet of Langerhands of the pancreas, Anteriorpituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes,Corticotropes, Intermediate pituitary cell, secretingmelanocyte-stimulating hormone; and Magnocellular neurosecretory cellssecreting oxytocin or vasopressin; Gut and respiratory tract cellssecreting serotonin, endorphin, somatostatin, gastrin, secretin,cholecystokinin, insulin, glucagon, bombesin; Thyroid gland cells suchas thyroid epithelial cell, parafollicular cell, Parathyroid glandcells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells,chromaffin cells secreting steroid hormones (mineralcorticoids and glucocorticoids), Leydig cell of testes secreting testosterone, Theca internacell of ovarian follicle secreting estrogen, Corpus luteum cell ofruptured ovarian follicle secreting progesterone, Granulosa luteincells, Theca lutein cells, Juxtaglomerular cell (renin secretion),Macula densa cell of kidney, Peripolar cell of kidney, and/or Mesangialcell of kidney.

Additionally or alternatively, one can treat at least one side of themembrane with Metabolism and storage cells such as Hepatocyte (livercell), White fat cell, Brown fat cell, Liver lipocyte. One can also useBarrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract)or Kidney cells such as Kidney glomerulus parietal cell, Kidneyglomerulus podocyte, Kidney proximal tubule brush border cell, Loop ofHenle thin segment cell, Kidney distal tubule cell, and/or Kidneycollecting duct cell.

Different geometries can be employed with dimensions related to thedifferent tissue types. For example, in one embodiment, relatively tallspacer open top chip dimensions are contemplated for the skin model,bronchial model, Kidney model and Gut model, i.e. chamber height between500 microns to 5 mm, chamber width 1 mm, chamber length 1.6 mm.

In another embodiment, relatively short spacer open top chip dimensionsare employed: chamber height between 100 to 500 microns, chamber width 1mm, chamber length 1.6 mm. These dimensions are better suited to theBrain barrier and Lung models.

An example of the importance of its application is the small airwaymodel: Small Airway cells feel the paracrine stimulation of neighborcells, which stimulate their fully differentiation. In the normal chipdesign cytokines are continuously flushed away from the epithelialcompartment by the constant flow, and this reduces or impedes epithelialcell differentiation. The presence of this porous matrix efficientlybuffers the effect of flow reducing or annul the effect of flow undercells.

The physical properties of the gels and fluids can vary (in addition tothe different geometries and dimensions for each of the different tissuetypes). For example, for the Skin model and bronchial model, arelatively high concentration collagen (8-11 mg/ml) is used. For theKidney model and Gut model, a 1:1 mixture of high concentrationcollagen:Matrigel is employed. For the Brain barrier and lung models, a1:1 low concentration (e.g. 3 mg/ml) of collagen/matrigel and/orfibronectin is employed. All in all, concentrations above 0.3 mg/ml arerequired to form gels. Preferred concentrations range between 3 mg/mland 10 mg/ml. However, concentrations above 5 mg/ml are particularlysuitable for use in the open top chip.

Not all of the organ models require a gel. Indeed, some organ chips areideally used without a gel (e.g. lung). When gels are used, more thanone gel layer can be employed. For example, hepatocytes can have a gelon both sides of the cells (e.g. a matrigel layer on top and a collagenlayer on the bottom. Importantly, the gel can have a variety ofthicknesses, including a thin (molecular) coating. In one embodiment,the coating is made with by inkjet printing.

Some cells do very well on patterned gels. For example, muscle cells dowell when they can deform to the surface. Indeed, in one embodiment, thepresent invention contemplates a gel pattern such that the sarcomeresalign.

Importantly, the present invention contemplates electrophysiologicalmeasurements in more than the blood brain barrier (BBB) model. Thepresent invention contemplates such measurements for muscle (whetherskeletal, cardiac or smooth muscle) cells.

DETAILED DESCRIPTION OF THE INVENTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings and will herein be described indetail preferred embodiments of the invention with the understandingthat the present disclosure is to be considered as an exemplification ofthe principles of the invention and is not intended to limit the broadembodiment of the invention to the embodiment illustrated. For purposesof the present detailed description, the singular includes the pluraland vice versa (unless specifically disclaimed); the word “or” shall beboth conjunctive and disjunctive; the word “all” means “any and all”;the word “any” means “any and all”; and the word “including” means“including without limitation.”

Those of ordinary skill in the art will realize that the followingdescription is illustrative only and is not intended to be in any waylimiting. Other embodiments will readily suggest themselves to suchskilled persons having the benefit of this disclosure. Reference willnow be made in detail to implementations of the example embodiments asillustrated in the accompanying drawings. The same or similar referenceindicators will be used throughout the drawings and the followingdescription to refer to the same or like items. It is understood thatthe phrase “an embodiment” encompasses more than one embodiment and isthus not limited to only one embodiment.

As used herein, the term “rigid” refers to a material that is stiff anddoes not stretch easily, or maintains very close to its original formafter a force or pressure has been applied to it. The term “elastomeric”as used herein refers to a material or a composite material that is notrigid as defined herein. An elastomeric material can be generallymoldable, extrudable, cuttable, machinable, castable, and/or curable,and can have an elastic property that enables the material to deform(e.g., stretching, expanding, contracting, retracting, compressing,twisting, and/or bending) when subjected to a mechanical force orpressure and partially or completely resume its original form orposition in the absence of the mechanical force or pressure. In someembodiments, the term “elastomeric” can also refer to a material that isflexible/stretchable but it does not resume its original form orposition after pressure has been applied to it and removed thereafter.The terms “elastomeric” and “flexible” are used interchangeably herein.

The functionality of cells, tissue types, organs, or organ-componentscan be implemented in one or more microfluidic devices or “chips” thatenable researchers to study these cells, tissue types, organs, ororgan-components outside of the body while mimicking much of the stimuliand environment that the tissue is exposed to in-vivo. In someembodiments, it is desirable to implement these microfluidic devicesinto interconnected components that can simulate the function of groupsof organs, organ-components, or tissue systems. In some cases it isdesirable to configure the microfluidic devices so that they can beeasily inserted and removed from an underlying fluidic system thatconnects to these devices in order to vary the simulated in-vivoconditions and organ systems (e.g., in situ conditions).

Many of the problems associated with earlier systems can be solved byproviding an open-top style microfluidic device that allows topicalaccess to one or more parts of the device or cells that it comprises.For example, the microfluidic device can include a removable cover, thatwhen removed, provides access to the cells of interest in themicrofluidic device. In some embodiments, the microfluidic devicesinclude systems that constrain fluids, cells, or biological componentsto desired area(s). The improved systems provide for more versatileexperimentation when using microfluidic devices, including improvedapplication of treatments being tested, improved seeding of additionalcells, and/or improved aerosol delivery for select tissue types. In apreferred embodiment, the open-top microfluidic device comprises a gelmatrix.

The present disclosure additionally relates to organ-on-chips (“OOCs”),such as fluidic devices comprising one or more cells types for thesimulation one or more of the function of organs or organ-components.Accordingly, the present disclosure additionally describes open-toporgan-on-chips that solve problems associated with earlier fluidicsystems. Without limitation, specific examples include models of skin,bronchial, and gut.

It is also desirable in some embodiments to provide access to regions ofa cell-culture device. For example, it can be desirable to providetopical access to cells to (i) apply topical treatments with liquid,gaseous, solid, semi-solid, or aerosolized reagents, (ii) obtain samplesand biopsies, or (iii) add additional cells or biological/chemicalcomponents.

The present disclosure relates to fluidic systems that include a fluidicdevice, such as a microfluidic device with an opening that providesdirect access to device regions or components (e.g. access to the gelregion, access to one or more cellular components, etc.). Although thepresent disclosure provides an embodiment wherein the opening is at thetop of the device (referred to herein with the term “open top”), thepresent invention contemplates other embodiments where the opening is inanother position on the device. For example, in one embodiment, theopening is on the bottom of the device. In another embodiment, theopening is on one or more of the sides of the device. In anotherembodiment, there is a combination of openings (e.g. top and sides, topand bottom, bottom and side, etc.).

While detailed discussion of the “open top” embodiment is providedherein, those of ordinary skill in the art will appreciate that manyembodiments of the “open top” embodiment apply similarly to open bottomembodiments, as well as open side embodiments or embodiments withopenings in any other regions or directions, or combinations thereof.Similarly, the device need not remain “open” throughout its use; rather,as several embodiments described herein illustrate, the device mayfurther comprise a cover or seal, which may be affixed reversibly orirreversibly. For example, removal of a removable cover creates anopening, while placement of the cover back on the device closes thedevice. The opening, and in particular the opening at the top, providesa number of advantages, for example, allowing (i) the creation of one ormore gel layers for simulating the application of topical treatments onthe cells, tissues, or organs, or (ii) the addition of chemical orbiological components such as the seeding of additional cell types forsimulating the function of tissue and organ systems. The presentdisclosure further relates to improvement in fluidic system(s) thatimprove the delivery of aerosols to simulate the function of tissue andorgan systems, such as simulated function of lung tissues.

Furthermore, the present disclosure contemplates improvements to fluidicsystems that include a fluidic device, such as a microfluidic devicewith an open-top region that reduces the impact of stress that can causethe delamination of tissue or related component(s) (e.g., such as a gellayer).

Improvements to microfluidic devices for simulating the function of atissue are contemplated by the present disclosure that include one ormore of an open-top microfluidic device with two or more chambers (e.g.,microchannels) separated by a membrane. In some embodiments, one or moreof the devices further comprises a gel in a chamber (e.g., microchannelor cavity) accessible through an opening, including but not limited toan open-top structure, of the microfluidic device. In some embodiments,the device further comprises a removable or permanent cover for themicrofluidic device where the cover optionally has a fluidic chamber ormicrochannel therein. Other desirable improvements that are contemplatedinclude a patterned gel in a microfluidic device.

The present disclosure further describes a method for culturing cells inopen-top devices. In some embodiments, the method comprises placing agel into an open-top structure. In some embodiments, the method furthercomprises patterning the gel using a shaping device, such as a patternedplunger stamp, a shaping stamp, or similar devices. In some embodiments,the method comprises permanently or reversibly applying a cover or othershaping device to the open-top.

The present disclosure further relates to the use of fluidic systemsthat include a fluidic device, such as a microfluidic device with anopen-top, to construct a model simulating the structure and/or one ormore functions of, for example, skin, bronchial, or gut. In someembodiments, these models benefit from the presence of gels, which forexample, can provide a mechanical, biochemical environment for one ormore cells types, augment the mass-transport characteristics, or providean additional compartment that may be used, for example, to house anadditional cell type (e.g. fibroblasts).

A system that provides for the use of a gel can be particularlydesirable for a skin model. For example, the current state-of-the-artskin model, the living skin equivalent (LSE), is a 3D gel, 2 mm to 3 mmthick, that is embedded with fibroblasts with differentiatedkeratinocytes on top of the gel. The actual thickness of the gel canrange from 0.1 mm to 5 mm. It is known that a 3D gel is preferred toproperly culture the fibroblasts that, in turn, enables keratinocytes tofully differentiate. An open-top architecture as described by someembodiments herein is desirable because it enables LSE-like and similarcultures of fibroblasts and keratinocytes, while further allowing theintroduction of an endothelial layer, the application of shear forces,and the application of stretching to create a more physiologicallyrelevant model. Each of these optional features, individually andcollectively, provides desirable improvements over currentstate-of-the-art LSE-like skin models.

Referring now to FIGS. 1 and 2 , one type of a microfluidic devicereferred to as an organ-on-chip (“OOC”) device 100 is illustrated thatmay be modified to include open-top embodiments that are described inmore detail later in this disclosure (see, e.g., FIGS. 3-5A-F and 8A -C-12 ). The OOC device 100 includes a body 112 that typically comprisesan upper body segment 101 and a lower body segment 103. The upper bodysegment 101 and the lower body segment 103 are typically made of apolymeric material, including, but not limited to, PDMS(poly-dimethylsiloxane), polycarbonate, polyethylene terephthalate,polystyrene, polypropylene, cyclo-olefin polymers, polyurethanes,fluoropolymers, styrene derivatives like styrene ethylene butylenestyrene (SEBS), or other polymer materials. The upper body segment 101,while illustrated with a first fluid inlet 117 and a second fluid inlet118, can be modified to include an open region 104 (not shown) tooptionally allow the application of a gel layer 150 (not shown) to amembrane 140 and optionally modified to exclude the illustrated firstfluid inlet 117 and/or second fluid inlet 118. A first fluid path for afirst fluid includes the first fluid inlet 117, a first seeding channel127, an upper microchannel 134, an exit channel 131, and then the firstfluid outlet 124. A second fluid path for a second fluid includes thesecond fluid inlet 118, a second seeding channel 128, a lowermicrochannel 136, an outlet channel 133, and then the second fluidoutlet 126.

Referring to FIG. 2 , a membrane 240 extends between the upper bodysegment 201 and the lower body segment 203. The membrane 240 ispreferably an inert, polymeric, micro-molded membrane having uniformlydistributed pores with sizes normally in the range of about 0.1 µm to 20µm, though other pore sizes are also contemplated. In some embodiments,the pore size is in the range of about 0.1 µm to 20 µm. The overalldimensions of the membrane 240 include any size that is compatible withor otherwise based on the dimensions of upper body segment 201 and lowerbody segment 103, such as about 0.05-100 mm (channel width) by about0.5-300 mm (channel length), though other overall dimensions are alsocontemplated. In some embodiments, the overall dimensions of themembrane 240 are about 1-100 mm (channel width) by about 1-100 mm(channel length). In one embodiment, the thickness of the membrane 240is generally in the range of about 5 µm to about 500 µm, and in someembodiments, the thickness is about 20-50 µm. In some embodiments, thethickness can be less than 1 µm or greater than 500 µm. It iscontemplated that the membrane 240 can be made of materials including,but not limited to poly-dimethylsiloxane (PDMS), polycarbonate,polyethylene terephthalate, styrene derivatives (e.g, styrene ethylenebutylene styrene, SEBS), fluoropolymers, and/or other elastomeric orrigid materials. Additionally, the membrane 240 can be made ofbiological materials including, but not limited to, polylactic acid,collagen, gelatin, cellulose and its derivatives,poly(lactic-co-glycolic acid), and/or comprise such materials inaddition to one or more polymeric materials. The membrane 240 separatesan upper microchannel 234 from the lower microchannel 236 in an activeregion 237, which includes a bilayer of cells in the illustratedembodiment. In some embodiments, a first cell layer 242 is adhered to afirst side of the membrane 240, and in some embodiments a second celllayer 244 is adhered to a second side of the membrane 240. The firstcell layer 242 may include the same type of cells as the second celllayer 244. Or, the first cell layer 242 may include a different type ofcell than the second cell layer 244. And, while a single layer of cellsis shown for the first cell layer 242 and the second cell layer 244,either the first cell layer 242, the second cell layer 244, or both mayinclude multiple cell layers or cells in a non-layer structure. Further,while the illustrated embodiment includes a bilayer of cells on themembrane 240, the membrane 240 may include only cells disposed on one ofits sides. Furthermore, while the illustrated embodiment includes cellsadherent to the membrane, cells on one or both sides may instead be notbe adherent to the membrane as drawn; rather, cells may be adherent onthe opposing chamber surface or embedded in a substrate. In someembodiments, the said substrate may be a gel.

The OOC device 100 is configured to simulate a biological function thattypically includes cellular communication between the first cell layer242 and the second cell layer 244, as would be experienced in-vivowithin organs, tissues, cells, etc. Depending on the application, themembrane 240 is designed to have a porosity to permit the migration ofcells, particulates, media, proteins, and/or chemicals between an uppermicrochannel 234 and a lower microchannel 36. The working fluids withinmicrochannels 234 and 236 may be the same fluid or different fluids. Asone example, as OOC device 100 simulating a lung may have air as a fluidin one channel and a fluid simulating blood in the other channel. Asanother example, when developing the cell layers 242 and 244 on themembrane 240, the working fluids may be a tissue-culturing fluid.Although it is not necessary to understand the mechanism of aninvention, it is believed that an organ-on-chip device offers utilityeven in the absence of cells on one side of the membrane, as theindependent perfusion on either side of the membrane can serve to bettersimulate the functions of mass-transport, shear forces, and otherembodiments of the biological environment.In one embodiment, the activeregion 237 defined by an upper microchannel 234 and a lower microchannel236 having lengths of about 0.1-10 cm, and widths of about 10-2000 µm.

The OOC device 100 preferably includes an optical window that permitsviewing of the fluids, media, particulates, etc. as they move across thefirst cell layer 242 and the second cell layer 244. Variousimage-gathering techniques, such as spectroscopy and microscopy, can beused to quantify and evaluate the effects of the fluid flow in an uppermicrochannel 234 and a lower microchannel 236, as well as cellularbehavior and cellular communication through the membrane 240. Moredetails on OOC devices can be found in, for example, U.S. Pat. No.8,647,861, and is incorporated by reference in its entirety. Consistentwith the disclosure in U.S. Pat. No. 8,647,861, in one preferredembodiment, the membrane 240 is capable of stretching and expanding inone or more planes to simulate functions of the physiological effects ofexpansion and contraction forces that are commonly experienced by cells.

Micro- and mesofluidic devices and membranes can be fabricated from orcoated with or otherwise produced from a variety of materials,including, but not limited to, plastics, glass, silicones, biologicalmaterials (e.g., gelatin, collagen, fibronectin, laminin, Matrigel®,chitosan, and others).

Turning now to FIGS. 3 through 12 various exemplary open-topmicrofluidic devices (e.g., open-top OOC devices) and components areillustrated that can be used for creating gel layers, such as for anopen-top skin-on-a-chip device or for creating gel layers for anopen-top OOC device for simulating other biological functions.

FIG. 3 illustrates an exploded perspective view of a cross-sectionthrough an exemplary open-top microfluidic device 300 (e.g., an open-topOOC device). Open-top microfluidic devices, such as an open-top OOCdevice, that allow access to the top of a chip offer several benefits.Topical treatment, such as for a skin-on-a-chip, can be applied directlythrough the open top to the tissue of interest. Topical treatments caninclude, for example, liquid, gas, gel, semi-solid, solid, particulateor aerosol. Furthermore, additional chemical or biological componentscan be added by means of the open top; as a particular example,additional cell types can be seeded within the open top of the device.Aerosol delivery, such as for a lung-tissue chip, is also contemplatedand can be completed through the open top, as well.

The microfluidic device 300 can optionally include a base 305, such as aglass slide, polymeric or metal support or a similar structure,optionally providing an optical window. The base 305 can support abottom structure 325 of the microfluidic device 300. The bottomstructure 325 defines a bottom chamber 336 connected to a bottom fluidicchannel 409 in the microfluidic device 300. Above the bottom structure325 is a membrane 340 having a membrane top side 348 and a membranebottom side 349. The membrane bottom side 349 is disposed on the topsurface of bottom structure 325 such that membrane bottom side 349 restsabove the bottom chamber 306. A top structure 320 is disposed on themembrane top side 348 of membrane 340 and defines an open region 304 forthe open-top microfluidic device 300 (e.g., the open-top chip). When thetop structure 320 is disposed on the membrane 340, it may be desirablethat all or substantially all of the open region 304 is bounded on thebottom by the membrane top side 348 of the membrane 340.

In some embodiments, the chamber of the top structure 320 can furtherinclude a top microfluidic cover fluidic channel 308 (not shown) such asa top microfluidic cover fluidic channel 508 (e.g., as illustrated inFIG. 5A). In FIGS. 5A - F, such a top microfluidic cover fluidic channel508 may permit perfusion of a top chamber 507, particularly while topchamber 507 is covered by an optional fluidic cover 510 (FIG. 5B). Insome embodiments, the present invention contemplates that embodimentsone or both of a bottom structure fluidic channel 509 and a topmicrofluidic cover fluidic channel 508 are microchannels. In someembodiments, the present invention further contemplates thatembodiments, an optional fluidic covers, such as fluidic cover 410 orfluidic cover 510 (see, FIGS. 4 and 5A - F, respectively) are disposedabove a top structure 520 and may further be in fluid communicationwith, and define a top chamber 507 and an open region 504. Although itis not necessary to understand the mechanism of an invention, it isbelieved that a fluidic cover, such as fluidic cover 410, may bedesigned for a one-time application (e.g. by means of bonding it inplace) or for subsequent removal.

An open region 304 in the open-top structure 320 may have any shape, butis preferably a notch. In one embodiment, the purpose of open region 304is believed to allow direct access to the membrane 340 or any matterdisposed above it, before, during, and/or after experimentation; suchaccess is not available in earlier closed microfluidic devices forsimulating tissues. While previous microfluidic devices, such as OOC,may have allowed for low viscosity fluids to be directed throughlimited-access channels to a membrane, such as illustrated in FIGS. 1and 2 , the open region 304 in top structure 320 additionally allows forthe placement of high viscosity gels, high viscosity fluids, solids,aerosols, and powders on an area of interest of membrane 340 (e.g., onthe membrane inclusive of a predetermined tissue culture).

Turning now to FIG. 4 , an exploded perspective view of an exemplaryopen-top microfluidic device 400 includes a fluidic cover 410. Themicrofluidic device 400 includes an optional base 405 that supports abottom structure 425. The bottom structure 425 defines a bottom chamber406. Above the bottom structure 425 and the bottom chamber 406 is aninterface region 488 that comprises a membrane 440. The membrane 440 isdisposed on the bottom structure 425 and above the bottom chamber 406. Atop structure 420 is disposed above the membrane 440 and includes a topchamber 407 with an open region 404. When the top structure 420 isdisposed on the membrane 440 during assembly of the device 400, it maybe desirable that all or substantially all of the open region 404 isbounded along the bottom by the membrane 440.

The fluidic cover 410 may be designed to permit the perfusion of theopen region 404 while the fluidic cover 410 is present. In someembodiments, the present invention contemplates that this configurationprovides an advantage over previous similar devices that allows theperfusion of the open region 404 by way of a top fluidic cover fluidicchannel 408 in the top structure 420. One of the benefits of including atop fluidic cover fluidic channel 408 in the fluidic cover 410 insteadof the top structure 420, is that cells, gel or other materials disposedin the open region 404 are not allowed to leak or spread into the topfluidic cover fluidic channel 408, where they may be undesirable. Forexample, cells in the top fluidic cover fluidic channel 408 will not beallowed to lie away from the active region 437 of membrane 440. Incontrast, by disposing a top fluidic cover fluidic channel 408 in thefluidic cover 410, a benefit is provided of topfluidic cover fluidicchannels 408 being absent when a fluidic cover 410 is removed, whichdisallows top fluidic cover fluidic channels 408 from being similarlyfilled with cells during seeding, as would happen with channels beingdirectly disposed in the top structure 420.

To minimize “leakage” of a substance of interest placed into an openregion 404 into areas where the substance is not desired, differentconfigurations of the open-top microfluidic device are contemplated. Forexample, a fluidic cover 410 can include a top chamber 407 (which may bea channel or part thereof) that substantially aligns with all or aportion of the open region 404 cover disposed in top structure 420. Thetop chamber 407 may optionally be hydraulically connected to one or morefluidic cover inlet ports 414 and/or fluidic cover outlet ports 416 (seealso, fluidic cover inlet port 514, FIG. 5D), which in some embodimentsmay be similar to the ports described for upper body segment 101 inFIGS. 1 and 2 . The presence of the top chamber 407 is especiallysignificant where the open region 404 is filled with a gel or othersubstance that impedes fluid flow. In such a case, the top chamber 407may be filled or perfused, enabling its contents to fluidically interactwith the substance in the open region 404. For example, if the openregion 404 holds a gel containing cells, flowing tissue-culture mediathrough the top chamber 407 (or even incubating this media without flow)would allow nutrients and reagents to be delivered to the cells, as wellas for waste products to be removed.

Through the use of a clamping device the fluidic cover 410 can bemechanically secured to the top structure 420 (e.g., see FIG. 5E) toprevent or minimize leakage of any fluidic substance of interest fromthe open region 404 of the open-top microfluidic device 400. Forexample, a spring-loaded clamp can be used to provide compression to abiocompatible polymer that uniformly seals the open region withoutadhesives. Such sealing can be further improved by including anelastomeric, pliable or soft material in at least one of the fluidiccover 410 or top structure 420; one with ordinary skill in the art willappreciate that many forms of gasketing and sealing may be applied here.An advantage of some embodiments that employ clamping is that theyfacilitate the application, removal and potentially the reapplication ofa lid or cover, which may desirably allow access to the open region 404after it was covered. Allowing access to an open region 404 of amicrofluidic device during experimentation can be useful, for example,in (i) the application of topical treatment, aerosol, additional cellsor other biological reagents, (ii) change of fluidic (e.g.tissue-culture media), (iii) sampling of fluidic or solid matter, or(iv) imaging using optical or other techniques. The option to repositionthe cover or apply a different cover further permits the continued useof the device (e.g. in a biological experiment). Alternatively, the lidor cover may be removed at the end of the device’s use to permitsampling that may be destructive, such as taking biopsies or otherwiseremoving samples, staining, fixing, or imaging.

In some embodiments, the present invention contemplates that a fluidiccover 410 can also, or alternatively, be bonded or otherwise disposedonto the top structure 420. For example, for fluidic or gas sealing, anadhesive membrane, laminate, film, or sheet can be used to temporarilyor permanently seal the open region at the interface between the topstructure that defines the open region and a removable cover. It is alsocontemplated that biocompatible polymer plugs or pistons can be used toseal off the open region. It is further contemplated that an open region404 of an open-top microfluidic device 400 can be simply covered (e.g.,similar to cell culture plates) with a cover or plate that limitsevaporation and improves sterile handling.

In embodimentsone embodiment, the present invention contemplates that atop structure 420 can be used with an open region 404, similar to awell, or with a removable fluidic cover 410 that may be akin to a flatlayer that seals the top structure 420. An optional configuration inFIG. 4 includes a top chamber 407 with a fluidic cover fluidic channel408 that can also introduce fluids into the microfluidic device such asfor perfusion or the introduction of other liquids into the system.

As discussed above, open-top microfluidic devices described herein offera number of advantages. For example, these devices allow the topicalapplication of compounds to a membrane, including compounds in the formor a gel or powder. The open-top design also allows for aerosol deliveryto effect a simulated function of a tissue directly from the top of themicrofluidic device. Furthermore, the open-top configuration allowsaccess to apply simulated effects of wounding to a tissue (e.g.,simulate effects of a burn or scratch on the skin or intestine) duringthe course of testing and the application of a treatment of interest allwithin the same microfluidic device and as part of the sameexperimentation cycle.

Furthermore, the open-top configurations described herein also allowdirect access to the epithelium, and thus, allow the ability to biopsy asample during testing. An open-top configuration also allows microscopyto be applied during use of a chip, such as the application of electronmicroscopy, high-magnification imaging methods, and laser-based imagingmethods by removing the top cover of the microfluidic device, whileoptionally maintaining the integrity of the experiment.

In some embodiments, it is desirable to simulate one or more functionsof lung, as such function simulations may be beneficial, for example, intesting compound transport and absorption through the lung, the effectof aerosolized or inhaled compounds, model lung disease, or otherwiseobserve lung response. In vitro models are known in the art, includingfor example a lung-on-a-chip microdevice disclosures in U.S. Pat. No.8,647,861, entitled, “Organ Mimic Device with Microchannels and Methodsof Use and Manufacturing Thereof,” and the small-airway on-a-chipmicrodevice disclosures in International Publication No. WO2015/0138034, entitled, “Low Shear Microfluidic Devices and Methods ofUse and Manufacturing Thereof,” both of which are hereby incorporated byreference herein in their entireties. A lung model that combines severalof desired features in the same model would be beneficial. Desiredfeatures include recapitulation of various elements of lung structureand morphology, and the ability to satisfactorily introduce compounds ormaterials as aerosols, fluidic access (e.g. to emulate blood or airflow), or mechanical forces. For example, a lung model is desirable thatminimizes loss of aerosol that can occur in delivery tubing and channelsand variation in the aerosol delivery along the length of the channel.According to some embodiments of the present disclosure, a lung modelthat includes one or more of such desired features can be constructed.For example, in one embodiment, a lung module is constructed using anopen-top device, such as that illustrated in FIG. 4 (whether employing afluidic cover 410, the optional cover of FIG. 3 , or no cover).Accordingly, lung epithelial cells (e.g. alveolar epithelial cells) canbe included or deposited within the open region 404. Optionally, thebottom structure 425 may include endothelial cells, motivated by thepresence of similar cells in the vasculature (e.g. capillary bed) of anin vivo lung. It is also contemplated that using the various embodimentsof open-top devices described herein, a lung model may be biologicallycultured or operated statically (i.e., for example, without continuousflow or with discrete exchanges of some portion of the liquid in thedevice) or under flow in either fluidic channels disposed in, forexample, the bottom structure 425, top structure 420, or cover 410, aswell as any combination of these modalities, which may optionally bevaried during operation (e.g. begin with discrete fluid exchanges, thenintroduce flow). In addition, the open region 404 or cell layers withinit may be cultured dry, under an air-liquid interface, or submerged,with this mode of culture optionally varied during use. For example,following the example of the lung-on-a-chip and small-airway-on-a-chipdevices, it may be desirable to begin lung culture under submergedconditions and transition to an air-liquid interface culture after somematuration period (e.g. ranging without limitation from 1 hour to 7days, or from 1 day to 14 days).

A particular advantage of the various open-top embodiments of thepresent disclosure is that aerosol may be delivered to the lung cells inthe open region, such as open region 404. In one exemplary embodiment,while operating the device without the optional cover (or by removingthe cover), aerosol can be delivered directly into the open region 404from above (or substantially above). The aerosol may be generated usingany of a variety of aerosol-generation techniques known in the art.Alternatively, an aerosol generation means may be included in a coverthat can be placed on top of the open region 404. A cover may beoptionally removed or exchanged during use; for example, anaerosol-generating cover may be applied when aerosol is desired andreplaced with a fluidic cover 410 when fluidic perfusion is desired. Insome embodiments, non-aerosol materials or samples can be applied tocells present in an open region, such as open region 404. This mayinclude, but are not limited to, materials or samples that are difficultto apply fluidically due to their properties, such as slurries, pastes,solids, or viscous fluids.

Referring now to FIGS. 5A-5F, multiple perspective views, includingadditional cross-sectional details through an exemplary open-topmicrofluidic device, are illustrated. The microfluidic device 500includes a membrane 540 disposed between a bottom structure 525 and atop structure 520. The bottom structure defines a bottom chamber 506,′and the top structure 520 includes a top chamber 506 that defines anopen region 504, of the microfluidic device 500. In some embodiments, itis desirable that the open region 504, includes a gel layer 550,comprising a porous volume, or another material for testing (e.g., anextracellular matrix or cells embedded in an extracellular matrix). Forexample, a gel layer 550 can include gels used in an organ-on-chip modelof the skin to house fibroblasts and to support a layer orkeratinocytes. In FIG. 5B, a gel layer 550 is introduced into the openregion 504 (see FIG. 5A) where the gel layer 550 is bounded on thebottom by membrane 540.

In some embodiments, a gel layer 550, or porous volume, is formed byinjecting one or more suitable precursors through one or more fluidicchannels included in the top structure 520 (such optional channels aredepicted in FIGS. 5A-5C). The one or more precursors can then be treatedas desired to form the gel or porous volume (e.g. UV light, chemicaltreatment, temperature treatment and/or incubation/waiting).Alternatively, the one or more precursors are in a final or near-finalform, where no additional active process is applied in order to generatethe gel or porous volume. While the approach of injecting the one ormore precursors through one or more fluidic channels included in the topstructure 520 can be adapted to permit consistent filling with gel orother porous volume, it typically results in the gel or porous volumefilling at least part of the said fluidic channels. This may beundesirable in some situations; for example, when dealing with a gelcontaining cells, it is desirable to limit the cells to the activeregion, lest they may not receive sufficient nutrient or biochemicalcues through the membrane.

Alternatively, the one of more precursors can be placed into the top ofthe open-top microfluidic device via the open region 504,. Such anapproach permits alternative embodiments that eliminate or limit spacesinto which the precursors may spread (e.g. one may avoid fluidicchannels included in the top structure 520 that are in fluidiccommunication with the open region 504,). In other embodiments, the oneor more precursors may be injected into the open region 504, by means ofa fluidic cover 510 that includes one or more fludic cover fluidicchannels 508 (an example is illustrated in FIG. 5D). Although suchembodiments may also result in a gel layer 550 formed in the fluidiccover fluidic channels 508, the fluidic cover 510 can be removed andoptionally replaced, removing at least part of the undesired material.

In some embodiments, it is desirable to limit or shape the gel volume orporous volume. For example, in an organ-on-chip model of the skin, it ismay be desirable to limit the thickness of a gel layer housingfibroblasts and supporting keratinocytes to a selected thickness.Without limitation, such thickness may be chosen from one or more of theranges of 10um to 200um, 100um to 1 mm, 0.5 mm to 5 mm, or 1 mm to 10mm. According to some embodiments, the extent of a gel layer 550, orporous volume, may be limited by a shaping device 559 (e.g., a shapingcover, a plunger 560 with a patterned base) that is present during theintroduction or formation of the gel or porous volume. This shapingdevice 559 may be removed and optionally replaced with a cover (e.g., afluidic cover 510) once a gel layer 550, or porous volume, has formed.The shaping device 559 may optionally include a chamber into which thegel or porous volume can conform, at least in part. Alternatively, ashaping device 559 may include one or more features that protrude intothe open region 504. FIG. 5C illustrates one type of a shaping devicewith features that protrude into the open region 504, which takes theform of a plunger stamp 560. In some embodiments, shaping devices areapplied before the introduction of one or more precursors for a gel orporous volume; for example, it could be introduced through fluidicchannels present in the top structure 520, a fluidic cover 510 or evenin the shaping device itself. In other embodiments, the one or moreprecursors are introduced before the application of the shaping device,whether through fluidic channels in the top structure 520 or fluidiccover 510, or introduced directly into the open region 504 (e.g. using asyringe, pipette or printing process). In such cases, the shaping devicemay optionally include features (e.g. holes, fluidic channels, cavities)designed to allow the capture of excess precursor. In some embodiments,the shaping device comprises a plurality of layers. For example, theshaping device may include a spacer layer used to define gel height anda flat cover to prevent the gel from passing the spacer’s height. All oronly a subset of these layers may be removed once the gel or porousvolume is defined, with the remaining layers (e.g. spacer layer)potentially remaining during device use or experimentation. In someembodiments, the top structure 520 may be removed after gel or porousvolume formation, and can be optionally replaced with a differentstructure or cover, that may or may not include an open region.

In one embodiment, the present invention contemplates a gel layer 2050comprising a plurality of gel micropillars 2053. FIG. 20A. For example,such gel micropillars 2053 may be arranged in symmetrical rows along thesurface of the gel layer 2050. FIG. 20B.

In one embodiment, the present invention contemplates a gel layer formedas a gel mesh 2054. FIG. 20A. For example, such a gel mesh 2054 may beformed as an insert within a top chamber 2006 or bottom chamber 2007.FIG. 20B.

In one embodiment, the present invention contemplates a shaping devicecomprising a plunger stamp 560 having a patterned surface 665 thatcreates a pattern in the gel or porous volume at a patterning interface555. Depending on the properties of the precursor materials (e.g.viscosity of the precursor and its change through curing), the shapingdevice may be removed before the gel or porous volume have fully formed.

FIG. 5D next illustrates a perspective view of the exemplary open-topmicrofluidic device of FIG. 5C after a plunger stamp 560 has beenremoved, including a patterned top surface 557 in the gel layer 550. Thepatterning includes depressions 558 in the patterned top surface 557 ofthe gel layer 550. The removable fluidic cover 510 can then be placedonto microfluidic device 500 such that top chamber 507 aligns withbottom chamber 506. An exemplary fluidic cover 510 can optionallyinclude fluidic channels. In the example illustrated, one of the fluidicchannels 508 extends from inlet hole 514 to the top chamber 507. Anoutlet fluidic chamber 515 ends at outlet hole 516 wherein the outletfluidic chamber 515 extends downwardly through the fluidic cover 510,and connects through an opening in the membrane 540, such that it isfluidically connected with chamber 506. The fluidic cover 510 may beremovable, and once removed it may be optionally reapplied or optionallyreplaced with a different cover.

FIG. 5E illustrates the exemplary open-top microfluidic device disposedwithin an exemplary clamping device 570. A clamping device 570 can bedesirable because no glue or bonding is needed to hold the variouslayers of the microfluidic device together. The clamping device appliedto an open-top microfluidic device optionally allows efficient removalof the removable cover during an experiment. The clamping device 570 forthe microfluidic device 500 can include an optional platform 585 forengaging a first side (e.g., the bottom side) of the microfluidic device500. In some embodiments, a plurality of elongated posts 590 can extendupwardly from the platform 585. A compression plate 580, which may flator may in some embodiments be uneven, is movably coupled to theplurality of elongated posts 590 such that the compression plate 580 isvertically slidable along the posts 590. In some embodiments, thecompression plate 580 engages a second side (e.g., the top side) of themicrofluidic device 500; in other embodiments, the compression plate 580retains a cover to the microfluidic device 500. A compression device 580provides compressive forces (e.g., see arrows 598) generally in adirection along the elongated posts 590. The compression device (e.g.,springs 595, elastomers, flextures, etc.) is operatively connected tothe compression plate 580 such that the compressive forces (e.g., seearrows 598) create a substantially uniform pressure on the second side(e.g., the top side) of the microfluidic device 500. Clamping devicecomponents can be made from different types of materials, including, butnot limited to, PMMA (e.g., acrylic), thermoplastics, thermosetpolymers, other polymer materials, metals, wood, glass, or ceramics. Inalternate embodiments, the compressive plate 580 may be held in placeusing a retention mechanism including, but not limited to, one or moreof screws, clips, tacky/sticky materials, other retention mechanismsknown in the art, or the combination of any of these mechanisms and/orthe aforementioned compression device. In some embodiments, a retentionmechanism retains a compressive plate 580 with respect to or against aplatform 585. In alternate embodiments, a retention mechanism retains acompression plate 580 with respect to or against a microfluidic device500. For example, screws can be used to fasten a compression plate 580against a microfluidic device 500 with a corresponding threaded holesincluded in a microfluidic device 500. As another example, a compressionplate 580 can include a clip feature (as a retention mechanism) thatclips into a suitable receiving feature of a microfluidic device. Insome embodiments, thae compression plate 580 comprises a cover for anopen area included in a microfluidic device 500. In other embodiments, acompression plate 580 retains an additional substrate that comprises acover for an open area included in a microfluidic device 500.

In some embodiments, a compression plate 580 may include at least oneaccess hole 581 that substantially aligns with a corresponding fluidport (e.g., inlet hole 514 or outlet hole 516) on a microfluidic device500 or an optional cover. In some embodiments, an access hole 581securely holds or comprises a fluid connector. Such a fluidic connectormay be beneficial in fluidically interfacing with a microfluidic device500 or optional cover without necessitating that a connector be includedin a microfluidic device 500 or optional cover.

A bottom surface area of the compression plate 580 may be greater orsmaller than a top surface area of the microfluidic device 500. In someembodiments, the platform 585 can have a width such that the compressionplate width is greater than the base width. The compression plate 580can further include finger nubs or tabs (not shown) protruding from acentral portion of the compression plate and extending beyond the basesuch that a compression plate width with the finger nubs is greater thanthe base width.

In embodiments that include elongated posts 590, it is contemplated thatthe plurality of elongated posts 590 are substantially parallel and thecompression plate 580 includes a plurality of apertures operative toallow an elongated post to pass through a respective aperture. Theplurality of elongated posts 590 supports the compression device (e.g.,springs 595). The compression device can include at least one spring 595extending around an outer boundary of at least one of the plurality ofelongated posts 590. In some embodiments, a compression plate 580comprises two springs 595 that provide a substantial uniform orequalized pressure to a compression plate where a compression plate is amobile part of the clamping device 570 that moves easily up and down (oralong other axes) to allow for easy manipulation of the clamped system.For example, the use of springs in a clamping device can be desirablebecause springs constants can provide for a wide range of translationdistances and forces and are versatile for situations where a clampingdevice may be positioned upside down for extended periods of time. Acompression plate 580 can be modified in area, shape, thickness, ormaterial.

Altough it is not necessary to understand the mechanism of an invention,it is believed that a maximum compressive force provided to amicrofluidic device by a clamping device is determined based on theforce required to create a fluidic seal between a compression plate 580or optional cover and a microfluidic device 500 (if such a seal isdesired), and a propensity for the collapse of microfluidic channels orchambers within the microfluidic device 500 or optional cover. In someembodiments, compressive forces provided can range from approximately 50Pa (approximately 0.007 psi) to approximately 400 kPa (approximately 58psi). In some embodiments, compressive forces provided can range fromapproximately 5 kPa (0.7 psi) to approximately 200 kPa (29 psi). In someembodiments, it is desirable that the amount of force or pressureapplied by a compression plate 580 to a microfluidic device 500 keep amicrofluidic device sealed or properly sandwiched between thecompression plate 580 and a platform 585 while not being so extreme asto cause the collapse of the microfluidic channels or to prevent desiredgas exchange.

A glass slide or other transparent window (e.g. made of PMMA,polycarbonate, sapphire, etc.) can be integrated into a clamping device570 to provide a rigid support for the microfluidic device whichimproves pressure distribution for flexible devices (such as those madefrom PDMS silicone) while enabling good optical access for macroscopic,visual, or microscopic imaging that may be desirable through viewingportions of the clamp system.

In one embodiment, the present invention contemplates that the describedclamping device can facilitate the use or positioning of the device inan upside down position. This can be a particularly desirable featureduring cell seeding of the underside of a chip membrane, commonly doneduring OOC co-culture. A compression device for the clamping device 570can include alternatives to springs or other aforementioned compressiondevices or retention mechanisms. For example, hydraulic or pneumaticcompression systems are contemplated. It is also contemplated that forrigid microfluidic devices compliant gaskets can be used. For example,the clamping device 570 can be fitted with a compliant gasket that has alevel of springiness to it rather than a spring itself. The compliantgasket materials would create an interface between the compression plate580 and the microfluidic device 500 or between an optional cover and themicrofluidic device 500. It is also contemplated that in someembodiments a compression device can utilize geometric shapes, such ascantilevered beams, as part of the device design to provide compressiveforce resulting from the case material flexure or compression. In someembodiments, the compressive force can also be provided with magnetic orelectromagnetic systems.

FIG. 5F illustrates a perspective view of an alternative exemplarycross-section through an open-top microfluidic device, similar to device500, with a bottom chamber 506 and open region 504 that are generallycircular from a top or bottom view perspective. Other embodiments caninclude an oval or football shape. Another exemplary feature includes amembrane 540 disposed between the bottom structure 525 and the topstructure 520, where the bottom structure defines the bottom chamber 506and the top structure defines the open region 504. The illustratedmembrane 540 limits passage between the channels (e.g., the open region504 and the bottom chamber 506) to a plurality of holes 541 that in someembodiments comprise less than the entire surface area of the membrane540 within the open region 504 and bottom chamber 506. The plurality ofholes 541 may include laser cut holes for passage of a gel, a porousvolume, or another material (e.g., an extracellular matrix or cellsembedded in an extracellular matrix) that has been disposed in the openregion for testing.

In some embodiments, an open-top microfluidic device allows for thedirect deposition of a matrix, for example a gel or a porous volume or abiodegradable polyester such as polycapolactone, into the open region oropen portion of an open-top microfluidic device. For example, agel-forming solution or precursor can be placed in a mold that isseparate from the microfluidic device. The mold can approximate theshape of the chamber or open region into which the gel volume will bedisposed for a desired experiment. Similar to setting a gel layer 550directly into the microfluidic device 500 (see FIGS. 5C-5D), a plungerstamp 560 is placed into the gel solution in the mold such that a bottomsurface of the plunger stamp is in contact with the gel solution in themold. The bottom surface of the plunger stamp includes the pattern offeatures 555 for imprinting into the gel solution. After the gelsolution has at least partially solidified, the plunger stamp is thenremoved from the gel solution, thereby creating a patterned gel surface557 to simulate the functions of a tissue microstructure. Once the gelhas solidified to the point where the gel will not break apart orotherwise separate, the patterned gel can be removed from the mold andbe inserted into the similarly shaped open region of the actualmicrofluidic device to be used for experimentation. Alternatively, or incombination, a suitably shaped volume or gel or porous volume can be cutto size, 3D printed or aggregated from smaller volumes, then disposedinto the open region. Further, a gel or porous volume can be 3D printeddirectly into the open region. In another related embodiment, a matrix(e.g., gel or porous volume) such as one formed as described for FIGS.5C-5D, can also be easily extracted (whether whole or in part) from thetop structure of an open-top microfluidic device, which providesbenefits by overcoming the problem of staining and high-resolutionimaging without having to stain an entire chip or having to reconstructcell-monolayers. The removal or insertion of a gel, porous materialand/or biological sample (e.g. biopsy, blood) to or from the open regionof an open-top microfluidic device is also desirable because it canallow access for testing of the subject tissue sample in themicrofluidic device and/or then the subsequent removal of the samplefrom an OOC device, which can then be used for other applications (e.g.,for implantation into a patient; additional analysis in another device).In an alternative embodiment, the gel or gel containing cells or tissuecan be patterned following culture of cells in the gel material.

In some embodiments of a microfluidic device, it is desirable to includea cover that comprises sensors or actuators. For example, a cover cancomprise one or more electrodes that can be used for measurement ofelectrical excitation. In some embodiments, such as where the devicecomprises a membrane (e.g., membrane 540), the one or more electrodescan be used to perform a measurement of trans-epithelial electricalresistance (TEER) for the membrane. It may also be desirable to includeone or more electrodes on the opposite side of the membrane 540. In someembodiments, the electrodes can be included in a bottom structure (e.g.,bottom structure 525). In some embodiments, the bottom structure can bean open bottom with bottom electrodes included on a bottom cover thatcan be brought into contact with the bottom structure. The bottom covermay support any of the features or variations discussed herein in thecontext of a top cover, including, for example, removability, fluidicchannels, multiple layers, clamping features, etc.

In some embodiments, it is desirable to simulate one or more functionsof skin, for example, in testing compound transport and absorptionthrough the skin, the effect of topical treatments on skin aging orhealing, modeling skin disease, or observing skin response such asdamage or sensitization. While in vitro skin models are known, such asliving skin equivalent (LSE), a skin model that combines severalfeatures in the same model would is desirable. For example, desirablefeatures can include recapitulation of various elements of skinstructure and morphology, topical access, fluidic access (e.g. toemulate blood flow), or mechanical forces. According to some embodimentsof the present invention, a skin model that includes one or more of suchdesired features can be constructed. In one exemplary embodiment, theskin model is constructed using the open-top device illustrated in FIG.5D. Accordingly, a gel layer 550, which may be considered to correspondto the skin’s dermal layer, is present in or introduced into (e.g. usingany of the aforementioned methods) the open region 504. Optionally, thegel layer 550 (or other matrix) may include embedded fibroblasts orrelated cells, motivated by the presence of similar cells in the dermallayer of in vivo skin. Furthermore, the gel layer 550 is topped bykeratinocytes, which are a primary cell type of the skin. Thekeratinocytes may, for example, be deposited on top of the gel layer 550(which can be done, for example, directly through the open top orintroduced fluidically through channels present in the top structure 520or cover 510) or present in the gel or other device component andallowed to biologically mature or develop into a cell layer at the topof the gel layer 550. Optionally, the bottom structure 525 includesendothelial cells, motivated by the presence of similar cells in thevasculature (e.g. capillary bed) of in vivo skin. Using variousembodiments of the open-top device described herein, the resulting skinmodel may be biologically cultured or operated statically (i.e., forexample, without continuous flow or with discrete exchanges of someportion of the liquid in the device) or under flow in either fluidicchannels disposed in the bottom structure 525 top structure 520 or cover510, as well as any combination of these modalities, which mayoptionally be varied during operation (e.g. begin with discrete fluidexchanges, then introduce flow). In addition, the open region 504 orcell layers within the open-top microfluidic device may be cultured dry,under an air-liquid interface, or submerged, with this mode of cultureoptionally varied during use. For example, following the example ofprior skin models such as the LSE, it may be desirable to beginkeratinocyte culture under submerged conditions and transition to anair-liquid interface culture after some maturation period (e.g. rangingwithout limitation from 1 hour to 3 days, or from 1 day to 14 days). Thegel layer 550 may comprise a biological or synthetic gel or other porousvolume, including for example, collagen I, collagen IV, fibronectin,elastin, laminin, gelatin, polyacrylamide, alginate, or Matrigel®.Collagen I in particular has been used by prior skin models, whereas itis known that elastin is present in in vivo skin, motivating its use inthe disclosed in vitro model.

In some embodiments, it can be similarly desirable to simulate one ormore functions of the intestine, for example, in testing compoundtransport and absorption through the intestine or its parts, the effectof treatments on intestine health or healing, modeling intestinaldisease, or observing intestinal response such as damage orsensitization. In vitro intestinal models are known in the art,including for example transwell-based systems or the gut-on-a-chipmicrodevice disclosures in U.S. Pat. Publication No. 2014/0038279,entitled “Cell Culture System,“ which is incorporated by referenceherein in its entirety. In some embodiments, it is desirable constructan intestinal model that combines several of the desired features in thesame model, including recapitulation of various elements of intestinalstructure and morphology, fluidic access (e.g. to emulate luminaltransport or blood flow), or mechanical forces. According to someembodiments of the present disclosure, an intestine model that includesone or more of such desired features can be constructed. In oneexemplary embodiment, the intestine model is constructed using theopen-top device illustrated in FIG. 5D. Accordingly, a gel layer 550, ispresent in or introduced into (e.g. using any of the aforementionedmethods) the open region 504. Furthermore, the gel layer 550 is toppedby intestinal epithelial cells. The intestinal epithelial cells may, forexample, be deposited on top of the gel layer 550 (which can be done,for example, directly through the open top or introduced fluidicallythrough channels present in the top structure 520 or cover 510) or bepresent in the gel or other device component and allowed to biologicallymature or develop into a cell layer at the top of the gel layer 550.Optionally, the bottom structure 525 includes endothelial cells,motivated by the presence of similar cells in the vasculature (e.g.capillary bed) of in vivo intestines. Optionally, the gel layer 550includes cells, for example, smooth muscle cells, neuronal cells,lymphatic cells or other cells types, cultures within the gel layer 550.Using various embodiments of the open-top device described herein, theresulting model may be biologically cultured or operated statically(i.e., for example, without continuous flow or with discrete exchangesof some portion of the liquid in the device) or under flow in eitherfluidic channels disposed in the bottom structure 525 top structure 520,or cover 510, as well as any combination of these modalities, which mayoptionally be varied during operation (e.g. begin with discrete fluidexchanges, then introduce flow). Although cells of the intestine aretypically cultured submerged, the open-top device also permits the openregion 504 or cell layers within it to be cultured dry or under anair-liquid interface, to simulate intestinal gas or various pathologies(e.g. swallowed air or gas presence with irritable bowel syndrome orlactose intolerance), or cultured with highly viscous or solidparticulate material (e.g., food, fecal matter, etc.) with the mode ofculture optionally varied during use. The gel layer 550 may comprise abiological or synthetic gel or porous volume, including for example,collagen I, collagen IV, fibronectin, elastin, laminin, gelatin,polyacrylamide, alginate, or Matrigel®.

It some embodiments, it can be similarly desirable to simulate one ormore functions of the small airway, for example, in testing compoundtransport and absorption through the airway or its parts, the effect oftreatments on airway health or healing, modeling airway disease, orobserving airway response such as damage or sensitization. In vitrosmall airway models are known in the art, including for example thesmall-airway on-a-chip microdevice disclosures in InternationalPublication No. WO 2015/0138034, entitled, “Low Shear MicrofluidicDevices and Methods of Use and Manufacturing Thereof,” which is herebyincorporated by reference herein in its entirety. According to someembodiments of the present disclosure, a small-airway model can beconstructed to include one or more desired features, including forexample fluidic access to airway and vasculature, several of thedifferentiated cell types found in the in vivo airway (e.g. ciliatedcells, mucus-producing cells), and immune response. In one exemplaryembodiment, the small-airway model is constructed using the open-topdevice illustrated in FIG. 5D.

Accordingly, a gel layer 550, is present in or introduced into (e.g.using any of the aforementioned methods) the open region 504.Furthermore, the gel layer 550 is topped by small-airway epithelialcells. The small-airway epithelial cells may, for example, be depositedon top of the gel layer 550 (which can be done, for example, directlythrough the open top or introduced fluidically through channels presentin the top structure 520 or cover 510). Optionally, the bottom structure525 includes endothelial cells, motivated by the presence of similarcells in the vasculature (e.g. capillary bed) of in vivo airway. Usingvarious embodiments of the open-top device described herein, theresulting model may be biologically cultured or operated statically(without continuous flow or with discrete exchanges of some portion ofthe liquid in the device) or under flow in either fluidic channelsdisposed in the bottom structure 525, top structure 520, or cover 510,as well as any combination of these modalities, which may optionally bevaried during operation (e.g. begin with discrete fluid exchanges, thenintroduce flow). In addition, the open region 504 or cell layers withinit may be cultured dry, under an air-liquid interface, or submerged,with this mode of culture optionally varied during use. The gel layer550 may comprise a biological or synthetic gel or porous volume,including for example, collagen I, collagen IV, fibronectin, elastin,laminin, gelatin, polyacrylamide, alginate, or Matrigel®.

In some embodiments, it is desirable to provide mechanical strain orforce to at least a portion of the fluidic device. In particular, it maybe desirable to apply mechanical force to at least some cells presentwithin the fluidic device. According to some embodiments, a mechanicalforce is applied to at least one portion of an open-top device byincorporating an actuation mechanism. In some embodiments, thisactuation mechanism can include one or more operational channels,similar to ones described by U.S. Pat. No. 8,647,861, which is herebyincorporated by reference herein in its entirety. Such operationalchannels can be evacuated or pressurized to cause the application offorce to a portion of the device, for example, a membrane separating atop and bottom fluidic channels. In this example, any cells present ontop or below the membrane may experience the mechanical force, leadingto a potential biological effect. In some embodiments, an open-topdevice is included in a system that additionally includes an actuationmechanism. In some embodiments, this actuation mechanism comprises asystem for mechanically engaging the open-top device and a system forapplying a stretch or compression force. A number of examples ofactuation systems included in a fluidic device or in systems thatinclude a fluidic device are described by International Application No.PCT/US2014/071570, filed Dec. 19, 2014, entitled “Organomimetic Devicesand Methods of Use and Manufacturing Thereof”, which is herebyincorporated by reference herein in its entirety. In one exemplaryembodiment, a system comprises an open-top device, a mechanical engagingdevice including one or more clamps or pins, and a mechanical actuationdevice including one or more electrical motors or pneumatic cylinders.According to one method to employ such a system, the open-top device isengaged with the mechanical engaging mechanism (e.g. by slipping the oneor more pins into corresponding holes included in the open-top device),and actuating said one or more electrical motors or pneumatic cylindersto apply a cyclical mechanical force on at least part of the open-topdevice.

Turning now to FIG. 6 , another exemplary shaping device 560 (in thiscase a plunger stamp 660) with a textured bottom surface 666 isillustrated for simulating biological conditions in an open-topmicrofluidic device (e.g., an open-top OOC device). The plunger stamp660 can be used in a similar manner as illustrated in FIGS. 5C and 5D.Plunger stamps can also be used to create gel layers of a definedthickness in the open region of an open-top microfluidic device. Thiscan be particularly beneficial where a separate section or layer may beneeded to introduce a dermal equivalent layer, such as a collagen plus afibroblast. A plunger stamp can also be beneficial for skin developmentin, for example, an open-top OOC device, by allowing the creation of athick gel layer (e.g., about 50 micrometers to about 10 millimeterthick, about 100 micrometers to about 1 millimeter thick), such as foran in vivo skin section. The plunger stamp can also be used inapplications where cells are embedded into a system, such as an ECM withthe introduction of cells into the matrix. Application of a plungerstamp to a gel in an open region of an open-top microfluidic device alsoallows for the embedding of fibroblasts into the gel layer.

Patterned surfaces created with a shaping device (e.g. plunger stamp)can provide for more accurate simulation of tissue or organcharacteristics, such as for skin tissue, small-airway tissue andintestine. For example, a gel layer for a skin model can be formed to beundulating, with the undulations mimicking features of in vivo papillaeor rete peg structures. Such structures are hallmarks of in vivo skinand can vary with skin health and age. Accordingly, the ability to formand control structures in the open-top chip that mimic in vivostructures is a beneficial embodiment of the disclosed open-topmicrofluidic systems. As a further example, patterning using a shapingdevice (e.g. plunger stamp 660) can be used to recreate structure in anintestinal model that mimic intestinal villi. Villi are understood to bea predominant cellular structure of the in vivo intestine, as amongstother things, they are believed to correspond to a villus-crypt axis ofcell differentiation. An ability to controllably form structures thatmimic villi in an intestinal model is another beneficial embodiment ofthe disclosed open-top microfluidic systems.

A type of pattern formed on a gel or porous volume may also determine ifdesired cell types will form in, or on, the said gel or porous volume.For example, adult keratinocyte cells may not differentiate and may dieif the geometry of the gel does not sufficiently simulate the cells’native environment. Using a patterned shaping device (e.g. patternedplunger stamp 660) that allows the imprinting of specific andsophisticated patterns (e.g., patterning and/or geometries simulatingthe native environment for cells being cultured) into the gel or porousvolume surface, a desirable micro-environment can be created that mayallow for cell survival and cell differentiation.

Turning now to FIG. 7 , an exemplary pattern for a plunger stamp 760 isillustrated. The plunger stamp 760 includes a patterned bottom surfacewith a plurality of papillae structures 767 that simulate the papillaestructure of the dermis, which when imprinted into the surface of a gellayer can be useful for differentiation of an adult skin equivalent.

In some embodiments, a gel layer is first placed into an open region ofa top structure of a microfluidic device or placed into a mold (e.g.,simulating an open region) followed by stamping of a gel surface with aplunger stamp. In other embodiments, a plunger stamp is first insertedinto an open region to a predetermined desired based on a desired gellayer thickness and a pre-polymerized gel with a lower-viscosity than inits final cured form is placed or allowed to flow into an open regionconfined by a plunger stamp, a membrane, and the sides of the openregion. A plunger stamp is dimensioned such that there are sufficienttolerances (e.g., gaps) between the side of a plunger stamp and the sidewalls of an open region (e.g., channel) so that a gel does not ooze orleak up the side of an open region when a pre-polymerized gel isimprinted with a patterned surface of a plunger stamp.

Referring now to FIGS. 8A - C -10 , an exemplary embodiment of anopen-top device 800 including round open regions 804 a, 804 b, 804 c isillustrated. The round open regions 804 a, 804 b, 804 c offer advantagesin the use of the device. For example, the device is amenable to biopsywith round biopsy punches typical for in vivo work, there is broad areaavailable for topical treatments or experimental procedures, and theymay provide a more isotropic biological environment than, for example,elongated sections. A more isotropic environment can be especiallybeneficial when present cells affect contractile or expansive forces, asis often the case with fibroblasts such as those present in thedermal-like layer of skin models. Although the depicted embodiments inFIGS. 8A - C are round, some of the aforementioned advantages also applyto other shapes, including for example ovals, shapes that inscribe roundsections, or other broad shapes.

FIGS. 8A - C-10 specifically illustrate stretchable embodiments of anopen-top microfluidic device 800. A stretchable open-top microfluidicdevice, such as the one illustrated in FIGS. 8A - C-10 can include openregions shaped in various ways including linear sections, althoughcircular, elliptical (e.g., from circular to a 1:2 ratio), or ovoid topregion seem to reduce the impact of tissue-induced stress that can leadto delamination of the tissue culture of interest (e.g., skin tissues).A stretchable device may allow for flow in a bottom fluidic layer thatis separated from a top fluidic layer by a permeable membrane (notshown), similar to the open-top microfluidic devices described for FIGS.3-5A - F. While the open-top microfluidic device 800 is described as astretchable device, it can be used with membranes other than stretchablemembranes (e.g., PDMS membranes) for applications where membrane stretchis not desired.

Turning to FIG. 8A, a top view of the exemplary assembled stretchableopen-top microfluidic device 800 is illustrated. The device 800 includesa top structure 820 that has three apertures therethrough which define aplurality of open-top open regions 804 a, 804 b, 804 c that may includea gel or porous volume. The open-top open regions may extend through theentire thickness of the top structure 820. As mentioned, mechanicalactuation can be effected in a variety of ways; in the illustratedexample, mechanical stretch is attained using one or more operatingchannels that on the perimeter of the open region. The top structure 820further includes a plurality of vacuum port pairs: i) 830 a, 832 a; ii)830 b, 832 b; and iii) 830 c, 832 c, that are in communication with theone or more vacuum chambers 837 a, 838 a, 837 b, 838 b and 837 c, 838c.The vacuum port pairs can be connected to a vacuum device that is usedto generate pressure differences that cause, for example, a membrane(not shown) to stretch (e.g., radially). Each open-top open region(e.g., 804 a) is illustrated as having two opposing vacuum ports (e.g.,830 a, 832 a), thereby forming a vacuum port pair. The illustratedconfiguration permits a mechanical stretch generated by the opposingvacuum chambers 837 a-c amd 838 a-c to apply a biaxial force on thedevice’s membrane active regions. Combined with the circular shape ofthe open regions, the device approximates isotropic stretch, which maybe desirable in the recapitulation of the biological mechanicalenvironment of some organs, including the skin. In alternativeembodiments, the shape of the open regions and vacuum chambers can bemodified to augment the directionality and non-isotropicity of thestretch. Moreover, devices that include a plurality of vacuum chamberscorresponding to one or more of the open regions allow the applicationof different pressures (including vacuum levels) permitting theselection of stretch directionality during use. The top structure 820further includes a plurality of bottom fluidic layer inlet ports 819 a,819 b, 819 c and outlet ports 822 a, 822 b, 822 c that allow for theintroduction and extraction of fluids (e.g., for perfusion) from theopen-top microfluidic device 800. FIG. 8B illustrates a perspective viewof the top structure of the exemplary stretchable open-top microfluidicdevice of FIG. 8A, and in particular shows how the open-top openregions, vacuum ports, vacuum chambers and bottom fluidic layer extendthrough the entire top structure 820. More or fewer (e.g., one, two,four, five or more) open-top open regions and related support featuresare contemplated.

Turning now to FIG. 8C, a perspective view of the bottom structure 825of the exemplary stretchable open-top microfluidic device 800 isillustrated. Similar to the previously described embodiments of anopen-top microfluidic device, a permeable membrane (not shown) isdisposed along the interface between the top structure 820 and thebottom structure 825. The bottom structure includes feeding channels 839a-c comprising a plurality of feeding channel wells (e.g., first feedingchannel well 835 a, second feeding channel well 835 b, third feedingchannel well 835 c) that align with open-top inlet and outlet ports(e.g., 514 and 516), respectively. A membrane (not shown) separates theopen-top open region (e.g., 804 a) from the feeding channels 839 a-c andfeeding channel wells (e.g., 835 a-c). It is contemplated that a gellayer in the device 800 can be formed on top of the membrane in theopen-top openings similar to what is described elsewhere herein (see,e.g., FIGS. 5C-5D).

FIGS. 9 and 10 illustrate exemplary perspective views of cross-sections9-9 and 10-10 through the stretchable open-top microfluidic device. ofFIG. 8A. With the top and bottom structure assembled, the bottom fluidiclayer inlet (e.g., 819 b) and outlet ports (e.g., 822 b) each extendthrough the membrane (not shown) such that the ports are eachhydraulically connected to feeding channels 839 a, 839 b, 839 c (e.g.,illustrated as long narrow channels) in the bottom structure 825 toallow for the circulation or introduction of fluids into the open-topmicrofluidic device. FIGS. 8A - C. Similarly, in FIG. 9 the vacuum portpairs (e.g., 930 a, 932 a; 930 b, 932 b; 930 c, 932 c) in the topstructure 920 each extend to vacuum chamber pairs: i) 937 a, 938 a; ii)937 b, 938 b; and iii) 937 c, 938 c formed by the interfacing of the topstructure 920 and bottom structure 925. The vacuum chambers are at leastpartially defined by a stretchable or deformable surface pairs such as1045 b and 1046 b that introduces pressure changes to actuate themembranes (not shown) at the interface of each of the open-top openings(e.g., 1004 b) and with the bottom wells (e.g., 1006 b.FIG. 10 .

Although it is not necessary to understand the mechanism of aninvention, it is believed that the presently disclosed vacuum chambersfunction to provide a pneumatic stretching of a membrane. For example,when placed under a vacuum, a first deformable surface 1645 and seconddeformable surface 1646 deflect towards each other as depicted by adeflection line 1647.

FIG. 16 . It is further believed that since the top portion of thedeformable surfaces are deflected at a greater angle than the bottomportion of the deformable surfaces, the induced stress is transferred tothe underlying membrane, thereby causing the membrane to stretch. A moredetailed depiction of deformable surfaces 1945, 1946 induce a deflection1947 that causes bending around the corner of the vacuum chamber wall,as shown by the change in position of the inner and outer dotted lines.FIG. 22 .

FIGS. 11 and 12 illustrate exemplary views of different bottom fluidicchannel configurations. In the embodiment illustrated in FIG. 11 , alower microchannel 1136 is split into a number of constituent channels1129. Although it is not necessary to understand the mechanism of aninvention, it is believed that the smaller diameter of these constituentchannels 1129, as compared to the diameter of a lower microchannel1136,may offer an advantage in terms of bubble/debris clearance and flowuniformity compared to the single wider channel. Alternatively, asillustrated in FIG. 12 , the lower microchannel 1236 can be take aspiral form 1251, or a serpentine or meandering form 2252 as illustratedin FIG. 22 . Altough it is not necessary to understand the mechanism ofan invention, it is believed that the configuration of FIG. 12 canprovide increased robustness in the face of bubbles and debris that maybe present, and can provide a more even flow rate than the lowermicrochannel 1136 design illustrated in FIG. 11 . However, the resultingchannel length of the lower microchannel 1236 configuration in FIG. 12is typically longer than in the lower microchannel 1136 designs similarto FIG. 11 , with a shorter microchannel length being advantageous insome applications. For example, the spiral lower microchannel 1251design illustrated in FIG. 12 first winds inwardly towards the center ofthe active region and the winds outwardly. An alternative design avoidsthe outward winding by flowing downward, either to a fluidic port or toan additional fluidic channel that may run underneath the spiralchannel.

In one embodiment, the present invention contemplates an open-topmicrofluidic device 1300 comprising at least two open regions 1304. Eachopen region 1304 may be configured with an inlet port 1314, an outletport 1316 and a vacuum port pair (1330, 1332).

In one embodiment, the present invention contemplates an open topmicrofluidic device 1400 comprising a top chamber 1407 or a bottomchamber 1406, said chambers having side walls 1443 where a plurality ofprojections 1413 protrude into a chamber lumen 1421. FIG. 14A and FIG.14B.

In one embodiment, the present invention contemplates an open-top chipdevice 1500 comprising at least two spiral lower microchannels 1551,wherein each of the microchannels are in fluidic communication with aninlet port 1519 and an outlet port 1522. FIG. 15 .

In one embodiment, the present invention contemplates an open-top chipdevice 1700 comprising: i) a first chamber 1763 and a second chamber1764, wherein each chamber is surrounded by a deformable surface 1745;and ii) at least two spiral microchannels 1751 located on the bottomsurface of the chambers, wherein each of the microchannels are influidic communication with an inlet port 1719 and an outlet port 1722and are respectively configured with a first vacuum port 1730 or asecond vacuum port 1732, such that each vacuum port is respectivelyconnected to a first vacuum chamber 1737 or a second vacuum chamber1738. FIG. 17 . An exploded view of the embodiment depicted FIG. 17shows an open-top chip device 1800, wherein a membrane 1840 residesbetween the bottom surface of the first chamber 1863 and the secondchamber 1864 and the at least two spiral microchannels 1851. FIG. 18 .

In some embodiments, the present invention contemplates an open-top chipdevice 2700 comprising at least two spiral lower microchannels 2751,wherein the microchannels are in fluidic communication with an inletport 2719 and an outlet port 2722. FIG. 27 . The spiral lowermicrochannel 2751 is also flanked by a vaccum port 2730 configured witha vacuum chamber 2737. A deformable surface 2745 is configured on theinside surface of the vacuum chamber 2737,

In some embodiments, the present invention contemplates an array device2811 comprising a plurality of open top chip devices 2800. Each of theopen top chip devices 2800 is configured with at least an open region2804 and flanked by an inlet port 2814 and an outlet port 2816, oralternatively, a first vacuum port 2730 and a second vacuum port 2732.FIG. 28 . An exploded view of an array device 3911 is provided showingthe open top chip devices 3900 in top structure 3920 and a bottomchamber 3906 in bottom structure 3925 with a membrane 3940 layeredbetween the top structure 3920 and bottom structure 3925. FIG. 39 .

Although it is not necessary to understand the mechanism of aninvention, it is believed that an array device comprising open top chipsrepresents a fundamental shift in architecture as compared toconventional “tissue-on-a-chip” designs. It is further believed thatthis array design facilitates multiplexing of 3D scaffold models forscaffold optimization. Furthermore, the array test platforms aredesigned to be compatible with existing 3D scaffold models in transwell.For example, array devices as contemplated herein are useful for 3Dscaffold models, ECM/gel optimization and tissue chips including, butnot limited to, skin, lung and intestine (e.g., gut). In one embodiment,an array device 2811 may have the following specifications:

Body Material PDMS Sylgard 184 Membrane Material PDMS Sylgard 184Dimensions Width 51.8 mm Length 50.8 mm Open-Top Chamber Dimensions TopChamber Diameter 6.3 mm Top Chamber Height 6 mm Top Channel Volume193.02 mm³ Top Culture Area 32.17 mm² Bottom Chamber Dimensions BottomChamber Diameter 5.4 mm Bottom Channel Height 1 mm Bottom Channel Volume22.90 mm³ Bottom Culture Area 22.90 mm² Membrane Dimensions PoreDiameter 7 µm Pore Spacing 40 µm(hexagonally packed) Thickness 50 µmCo-culture Area 22.9 mm² Minimum Imaging Distance (top of membrane) 2 mm

Additional exemplary embodiments of open-top microfluidic devices, suchas the devices discussed above in FIGS. 1-12 , are now describedfurther. In some embodiments, the dimensions of the top area of the openregion in a top structure for a chip can range from about 0.1 to about17 millimeters (or 1 to about 7 millimeters) along in the narrowestdimension. In some embodiments, the dimensions range from about 0.5 toabout 200 or more millimeters (or about 0.5 to about 20 millimeters).The lower end of the range of the narrowest dimension of the open regionis also desirably sized to allow accessibility to the region forpipettes or syringes that are used to place, for example cell culturesor gel materials. The open region can be sized to limit any capillaryaction, which may be undesirable in some applications (capillary actionmay nevertheless be desirable in other applications). It is furtherdesirable in some applications for the upper range of the open regiondimensions to be sized to maintain accuracy in the flow distribution forthe bottom channel across the cell culture area.

In some embodiments, the depth of the open region (e.g., measuringvertically upward in the open region from the interface of the topstructure with the membrane) can vary from about 0.1 to about 20millimeters (or about 1 to about 5 millimeters). In some embodiments, anadditional well or spacer may be added to increase the well volume ofthe open region, such as where the full depth of the open region iscompletely filled. It is contemplated that aspect ratios of thedimensions for the top area to the depth of the open region in someapplications should range from about 1 to above 100, or in someapplications from about less than 0.01 to 2.

In some embodiments, it is desirable to have different geometries forthe open region based on the type of tissue that is subject toexperimentation. For example, certain types of tissue, such as skin, arehighly contractile during culturing. When placed into high-aspect ratio(e.g., 16 millimeters by 1 millimeter) channels, delamination of thetissue can occur along the narrow dimension. However, an open regionthat has a circular (e.g., open region 804 a) provides radial symmetrythat can allow tissue to shrink uniformly and not move out of plane. Awider channel geometry that minimizes edge effects can also bebeneficial for other organ systems that may require multiple layers,such as the blood-brain barrier, airways, or digestive tract, becausethe layers can be more easily formed by the sequential deposition ofthin gel or cellular tissue layers, which is difficult to do in closedchannels or chambers. In some embodiments, the geometry of the openregion is something different than the rectangles or circles illustratedin the exemplary embodiments of FIGS. 5A - F and 8A - C. For example, atriangular or star geometry can be used to look at the effects of cellcrowding or diffusion of signaling molecules as affected by geometry. Inanother example, a “FIGS. -8A - C” shape can be beneficial for analyzingthe interaction between two three-dimensional cultures

For fluidic channel(s) disposed in the top structure of an open-topdevice that might be used for skin, bronchial, or gut tissuesimulations, the geometry and dimensions for the open region of achamber can include a channel-type geometry with a channel heightranging, for example, from about 0.02 millimeters to about 10millimeters, a channel width of about 0.05 millimeter to 20 millimeter,and a channel length of about 0.5 millimeters to about 300 millimeters.In some embodiments, the geometry and dimensions for the open region ofa top chamber can include a channel-type shape with a height ranging,for example, from about 0.02 millimeters to about 10 millimeter and afluidic cover fluidic channel width of about 0.05 millimeter to 20millimeter. The base or bottom chamber can also have a channel-typeshape with a height ranging, for example, from about 0.02 millimeters toabout 10 millimeter. For an optional top structure 420 that might beused for brain-barrier and lung tissue simulations, the geometry anddimensions for the top structure, for example, include a height of about0.05 millimeters to about 5 millimeter. A taller top structure spacer inan open-top microfluidic device is often used for simulations wherethree-dimensionality is desirable, such as where fibroblast or othercells are embedded in the gel layer for the formation of, for example, adermal layer. A shorter top structure spacer in an open-top microfluidicdevice can be used, for example, for simulations where two- orthree-dimensionality is desired, such as for small airway simulationswhere small airway cells feel the paracrine stimulation of neighborcells, which stimulates their full differentiation.

Various tissue types are contemplated for testing in an open-topmicrofluidic device (e.g., an open-top OOC device), such as skin,small-airway, and alveolar tissues. However, open-top microfluidicdevices can also accommodate other types of tissues, as well, includingother epithelial tissues.

The properties of gels or porous volumes that can be used for anopen-top microfluidic device can vary and the properties will oftendepend on the different tissue type that is being tested. For example,different tissue types or specific models may employ differentextracellular matrix proteins (ECMs) and ECM mixtures (for example,collagen I, collagen IV, Matrigel®, laminin, fibronectin, gelatin,elastin, etc., and combinations thereof). Additionally, some embodimentsmay employ synthetic polymer gels (e.g. polyacrylamide, polyvinylalcohol, etc.) or various other gels known in the art (e.g. agarose,alginate, etc.) alone, in mixture, or in combinations with ECMs.Similarly, porous volumes used for an open-top microfluidic device mayinclude a variety of open-cell foams, for example, expandedpolyurethane, expanded polystyrene, expanded cellulose, expandedpolylactic acid, etc. Without being bound by example, for the simulationof a skin or bronchial tissue, the gel can have a higher concentrationof collagen, roughly at about 1 to about 11 milligrams per milliliter ofgel. For the simulation of gut tissue function, one exemplary embodimentcontemplates a gel with a 1:1 ratio of a high concentration collagen toan ECM such as the Corning® Matrigel® matrix available from Corning LifeSciences, is desirable. For the simulation of alveolar tissue function,one exemplary embodiment contemplates a gel with a 1:1 ratio of a lowconcentration of collagen (e.g., about 3 milligrams per milliliter ofgel) to ECM, such as the Corning® Matrigel® matrix or fibronectin, isdesirable. It is contemplated in one embodiment that extracellularmatrices or other gel precursors that form gels with concentrations ofabove 5 milligrams per milliliter of gel, or ranging from about 3 toabout 15 milligrams per milliliter of gel, or ranging from about 0.2 to4 milligrams, can be used in the open-top microfluidic devices describedherein. Moreover, cross-linking agents such as, but not limited to,transglutaminase, glutaraldehyde, bis(sulfosuccinimidyl)suberate, andmany other cross-linkers known in the art, can be used to increase gelstiffness and optionally lower gel concentration. With the use ofcross-linkers, it is contemplated that extracellular matrices or othergel precursors that form gels with concentrations ranging from about0.05 to 5 milligrams per milliliter of gel, or ranging from about 1 to10 milligrams per milliliter of gel, can be used in the open-topmicrofluidic devices described herein.

While the described open-top microfluidic devices, including open-topOOC devices, are compatible with standard microfluidic fluids havingrelatively low viscosities (e.g., about 1 to about 10 centipoise orless), the open-top devices are well-suited for high viscosity solutionsand gels having a viscosity equal to or greater than 10 centipoise alongwith being well-suited for the polymerization of gels in situ for laterremoval from the microfluidic device and other manipulation of the gel.For example, collagen gels with a high protein content (e.g., 3milligrams per milliliter) can be directly pipetted into the open topsand gelled in place without shearing cells or requiring high pressureactuation. For drug testing applications, creams and similarhigh-viscosity materials can be spread directly on the tissue using theopen tops to test compounds in the final formulations rather thandissolved drugs alone. Thick gels layers can also be easily generatedfor three-dimensional culture applications with the potential forproviding mechanical stretch. Other desirable embodiments of open-topmicrofluidic devices include the open tops are readily compatible withaerosol and other particulate (e.g., liquid or solid) delivery whileminimizing loss, which allows for enabling high dosing accuracy. Becausethe particles can be delivered directly to the tissue, there is minimalloss due to adsorption to other surfaces, such as tubing andmicrochannels.

In some embodiments, the gel layer described in the above embodimentsdoes not need to be patterned. It is also contemplated that a gel orother material suitable for growing tissues can be patterned externally,shaped to fit the open region of the channel or chamber of the topstructure, and subsequently inserted into the open-top microfluidicdevice for cell culture. The gel or other material could also be a largesheet that is compressed using the spring loaded clamps with the twochambers or channels on either side of the gel or other material, wherethe gel or other material acts as a membrane in the open-topmicrofluidic device. The externally-prepared material can includebiological tissue such as a biopsy from a patient or small piece ofartificial tissue prior to implantation, and thus allow the performanceof assays on tissue to determine drug response, tissue quality, andother factors. It is further contemplated that the gel or a similarmaterial from the open-top microfluidic device can be extracted via theopen top and used for in vivo applications. For example, themicrofluidic device could be used to pattern and mature the tissue priorto implantation.

Numerous skin substitutes are commercially available, such as epidermalsubstitutes, dermal substitutes, and bilayer substitutes. These can beemployed together with the devices, layered structures and methodsdescribed above.

PREFERRED EMBODIMENTS A. Blood Brain Barrier

Brain microvascular endothelial cells (BMEC) are interconnected byspecific junctional proteins forming a highly regulated barrierseparating blood and the central nervous system (CNS), the so-calledblood-brain-barrier (BBB). Together with other cell-types such asastrocytes or pericytes, they form the neurovascular unit (NVU), whichspecifically regulates the interchange of fluids, molecules and cellsbetween the peripheral blood and the CNS.

The blood-brain barrier is of major clinical relevance becausedysfunction of the blood-brain barrier leads to degeneration of theneurovascular unit, and also because drugs that are supposed to treatneurological disorders often fail to permeate the blood-brain barrier.Due to its importance in disease and medical treatment, it would behighly advantageous to have a predictive model of the human blood-brainbarrier that recapitulates aspects of the cerebral endothelialmicroenvironment in a controlled way.

In one embodiment, the present invention contemplates a layeredstructure comprising i) fluidic channels covered by ii) a porousmembrane, said membrane comprising iii) a layer of brain microvascularendothelial cells and said membrane positioned below iv) a gel matrix(or other porous volume). The present invention contemplates, in oneembodiment, living neuronal cells (e.g. neurons, astrocytes, pericytes,etc.) on, in or under the gel matrix. It is preferred that some portionof the device can be opened for access to these cells. In oneembodiment, the device comprises a removable top. The gel can bepatterned to control the positioning and/or orientation of the cells orportions thereof. For example, the pattern on the gel matrix can directneurite growth for neurons seeded on the patterned surface.

B. Transepithelial Electric Resistance

There are many ways to evaluate the integrity and physiology of an invitro system that mimics the blood brain barrier. Two of the most commonmethods are Transepithelial Electric Resistance (TEER) and LuciferYellow (LY) rejection. Lucifer Yellow (LY) travels across cellmonolayers only through passive paracellular diffusion (through spacesbetween cells) and has low permeability. Therefore it is considerablyimpeded in passing across cell monolayers with tight junctions.Permeability (Papp) for LY of ≤ 5 to 12 nm/s has been reported to beindicative of well-established monolayers. One of skill in the art wouldunderstand that manipulations should be performed using aseptictechniques in order for the cells to remain in culture withoutcontamination. TEER measures the resistance to pass current across oneor more cell layers on a membrane. Specifically, this electricalresistance is a direct measurement of the resistance of the cellmonolayer to the transport of ions. The measurement may be affected bythe pore size and density of the membrane, but it aims to ascertain celland/or tissue properties. The TEER value is considered a good measure ofthe integrity of the cell monolayer.

For TEER measurements, an embodiment is contemplated wherein a layeredstructure or microfluidic device 2300 has a top electrode 2371 and abottom electtrode 2372 configured for measuring the electrophysiology ofsaid brain microvascular endothelial cells. FIG. 23 In one embodiment,the top electrode 2371 is a chromium/gold (Cr—Au) electrode. In oneembodiment, the bottom electrode 2372 is a chromium/gold (Cr—Au)electrode.

However, it is not intended that the present invention be limited toonly TEER measurements. In one embodiment, the present inventioncontemplates a method of testing, comprising 1) providing a layered TEERmicrofluidic device 2300 comprising i) a bottom structure 2325comprising at least one upper microfluidic channel 2334 covered by ii) aporous membrane 2340, said membrane comprising iii) a layer of brainmicrovascular endothelial cells in contact with said at least one uppermicrofluidic channel, said membrane position below iv) a gel matrix (orother porous volume), said gel matrix (preferably) under a removablecover; and 2) measuring the electrophysiology of said brainmicrovascular endothelial cells. In one embodiment, the porous membrane2340 is covered by a top structure 2320. In one embodiment, the layeredTEER microfluidic device 2300 further comprises a top clamp 2379 and abottom clamp 2384, wherein said top clamp 2379 has at least one accesshole 2381. In one embodiment, the at least one access hole 2381 isconfigured to align with a port adapter 2383. In some embodiments, aglass slide 2382 is placed between the bottom electrode 2372 and thebottom clamp 2384. In one embodiment, the top clamp 2379 comprises alasercut acrylic material. In one embodiment, the port adapter 2383comprises a cast PDMS material. In one embodiment, the top electrode2371 comprises a lasercut PET material. In one embodiment, the bottomelectrode 2372 comprises a lasercut PET material. In one embodiment, thetop structure 2320 comprises an open-top channel gasket having a castPDMS material. In one embodiment, the bottom structure 2325 comprises anopen-bottom channel gasket having a spincoated and lasercut PDMSmaterial. In one embodiment, the bottom clamp 2384 comprises a 3Dprinted ABS plastic material. Although not limiting, the top clamp 2379and bottom clamp 2384 may be attached with M4 screws 2386 and M4 nuts2387. Although it is not necessary to understand the mechanism of aninvention, it is believed that a TEER microfluidic device is clampedbecause the various layered components described above would bedifficult to glue (e.g., bonding). It is further believed that a clampfacilitates an ability to open the device and have direct access tocells for patch-clamp measurements. Alternatively, if this openablefeature is not desired, the device layers can be bonded together. Afully assembled layered TEER chip 2400 between a top clamp 479 andbottom clamp 2384 is presented in FIG. 24 .

A variety of techniques are contemplated including but not limited tousing a multi-electrode array or patch clamping. In one embodiment, thepresent invention contemplates an “open top” design that allows forpatch clamping through the opening. For example, an open-top patch clamplayered TEER microfluidic device 2500 may comprise an optional topmicrofluidic cover 2510 comprising an open region 2504, an optional topmicrofluidic cover fluidic channel 2508 and inlet port 2514, wherein theopen region 2504 provides access to an open-top channel gasket 2573. Inone embodiment, the TEER microfluidic subassembly device 2500 comprisesan open-top channel gasket 2573 having at least one upper microchannel2534 in fluid communication with at least one upper microchannel well2523. A porous membrane 2540 is placed between the open-top channelgasket 2573 and an open-bottom channel gasket 2574, wherein theopen-bottom channel gasket 2574 comprises at least one lowermicrochannel 2536. A bottom electrode 2572 is placed underneath theopen-top channel gasket/porous membrane/open-bottom channel gasketlayered stack. In one embodiment, the bottom electrode 2572 is achromium/gold electrode. FIG. 25

An open-top TEER microfluidic subassembly patch clamp device 2600 may beexposed to allow access with a micro-manipulator 2661. FIG. 26 . Forexample, a micromanipulator arm 2661 my be placed directly within anupper microchannel 2634. Although it is not necessary to understand themechanism of an invention, it is believed that the micromanipulator arm2661 may, for example, add reagents, remove a fluid sample, add cellsand/or remove cells. This allows the configuration of the patch clampdevice 2600 to interchangeably go between a flow configuration (e.g.,where the upper microchannel 2634 is not exposed) and an openconfiguration (e.g., where the upper microchannel 2634 is exposed).

C. Stretchable Open Top Chips

In one embodiment, the present invention contemplates a stretchable opentop chip device 2900 comprising at least one spiral microchannel 2951configured with at least one fluid inlet 2917 and at least one fluidoutlet 2924. FIG. 29A. In one embodiment, the microfluidic chip device2900 further comprises a upper microchannel with a circular chamber 2956configured with a first fluid or gas port pair 2975 and second fluid orgas port pair 2976, a first vacuum port 2930 connected to a first vacuumchamber 2937 and a second vacuum port 2932 connected to a second vacuumchamber 2938, wherein the vacuum chambers are proximally configuredaround the spiral microchannel. In one embodiment, the uppermicrochannel with a circular chamber 2956 is positioned above the spiralmicrochannel 2951. FIG. 29B.

Although it is not necessary to understand the mechanism of an inventionit is believed that the strechable open top chip design represents afundamental shift in architecture as compared to conventional“tissue-on-a-chip” designs. It is further believed that the open topdesign is compatible with 3D scaffold models. For example, an open topchip design may include, but is not limited to, three layers exemplifiedby a bottom channel, a middle chamber and a top channel. In oneembodiment, the bottom channel layout may be spiral in shape in order tofit within the circular shape of the chamber. In another embodiment, thetop channel allows for the ability to run media solutions orhumidity-controlled gases (e.g., for example, air and/or oxygen-carbondioxide mixtures such as 95% O₂/ 5% CO₂) to prevent gel evaporation. Inanother embodiment, the membrane is porous to facilitate cell-to-cellcommunication. Other embodiments provide a vacuum channel design thatprovides a mechanical stretch to the entire 3D scaffold thickness.

Furthermore, the open top strechable chips as contemplated herein areuseful for biological interfaces, co-cultures, multiple cell typecultures, tissue streching, 3D scaffold models, micro-patterning andtissue chips including, but not limited to, skin, lung and intestine(e.g., gut). In one embodiment, an open top strechable device may havethe following specifications:

Body Material PDMS Sylgard 184 Membrane Material PDMS Sylgard 184Dimensions Width 15.87 mm Length 35.87 mm Height 6.0 mm Top ChannelDimensions Top Channel Height 200 µm Top Chamber Diameter 5.70 mm TopChamber Dimensions Top Chamber Diameter 5.70 mm Top Chamber Height 4.00mm Top Channel Volume 102.07 mm³ Top Culture Area 25.52 mm² BottomChannel Dimensions Bottom Channel Width 600 µm Bottom Channel Height 400µm Bottom Channel Volume 5.446 mm³ Bottom Culture Area 13.6 mm² MembraneDimensions Pore Diameter 7.0 µm Pore Spacing 40 µm (hexagonally packed)Thickness 50 µm Minimum Imaging Distance (top of membrane) 850 mm

In one embodiment, the present invention contemplates a stretchable opentop chip device 3000 comprising: i) a fluidic cover 3010 comprising anupper microchannel with a circular chamber 3056 configured with a firstfluid or gas port pair 3075 and second fluid or gas port pair 3076; afluid inlet port 3014, a fluid outlet port 3016, a first vacuum port3030 and a second vacuum port 3032; ii) a top structure 3020 comprisinga chamber 3063, a first vacuum chamber 3037 connected to the firstvacuum port 3030, and a second vacuum chamber 3038, connected to thesecond vacuum port 3032, wherein the upper microchannel with a circularchamber 3056 overlays the top surface of the chamber 3063; and iii) abottom structure 3025 comprising a spiral microchannel 3051 comprisingan inlet well 3068 connected to the fluid inlet port 3014 and an outletwell 3069 connected to the fluid outlet port 3016, wherein a membrane3040 is layered between the top struture 3020 and bottom structure 3025.FIG. 30 .

In one embodiment, the present invention contemplates a stretchable opentop chip device 3100 comprising a chamber 3163 comprising an epithelialregion 3177 and a dermal region 3178. In one embodiment, the epithelialregion comprises an epithelial cell layer. In one embodiment, the dermalregion comprises a dermal cell layer, wherein said epithelial cell layeradheres to the surface of the dermal cell layer. In one embodiment, thedevice further comprises a spiral microchannel 3151 in fluidcommunication with a fluid inlet port 3114, wherein the microchannelcomprises a plurality of vascular cells. In one embodiment, a membrane3140 is placed between the chamber dermal cell layer and themicrochannel plurality of vascular cells. In one embodiment, the devicefurther comprises an upper microchannel with a circular chamber 3156connected to a fluid or gas port pair 3175. In one embodiment, thedevice further comprises a first vacuum port 3130 connected to a firstvacuum chamber 3137 and a second vacuum port 3132 connected to a secondvacuum chamber 3138. In one embodiment, the membrane 3140 comprises aPDMS membrane comprising a plurality of pores 3141, wherein said pores3141 are approximately 50 µm thick, approximately 7 µm in diameter,packed as 40 µm hexagons, wherein each pore has a surface area ofapproximately 0.32 cm². Although it is not necessary to understand themechanism of an invention, it is believed that the pore surface areacontacts a gel layer (if present). FIGS. 31A and 31B.

In one embodiment, the present invention contemplates a stretchable opentop chip device 3200 comprising: i) a fluidic cover 3210 comprising anupper microchannel with a circular chamber 3256 configured with a firstfluid or gas port pair 3275 and second fluid or gas port pair 3276; afluid inlet port 3214, a fluid outlet port 3216, a first vacuum port3230 and a second vacuum port 3232; ii) a top structure 3220 comprisinga chamber 3263, a first vacuum chamber 3237 connected to the firstvacuum port 3230, and a second vacuum chamber 3238, connected to thesecond vacuum port 3232, wherein the upper microchannel with a circularchamber 3256 seals with the top surface of the chamber 3263; and iii) abottom structure 3225 layered underneath said top structure 3220. FIG.32 .

FIGS. 33A and 33B illustrate exploded views of two embodiments of astretchable open top chip device comprising: i) a fluidic cover 3310comprising an upper microchannel with a circular chamber 3356 configuredwith a first fluid or gas port pair 3375 and second fluid or gas portpair 3376; a fluid inlet port 3314, a fluid outlet port 3316, a firstvacuum port 3330 and a second vacuum port 3332; ii) a top structure 3320comprising a chamber 3363, a first vacuum chamber 3337 connected to thefirst vacuum port 3330, and a second vacuum chamber 3338, connected tothe second vacuum port 3332, wherein the upper microchannel with acircular chamber 3356 overlays the top surface of the chamber 3363 and afirst membrane 3340 layered between the fluidic cover 3310 and the topstructure 3320; and iii) a bottom structure 3325 layered underneath saidtop structure 3220, wherein a second membrane 3340 is layered betweenthe bottom structure 3325 and the top structure 3320. FIGS. 33A - B.

FIG. 34A illustrates an assembled top view of a stretchable open topchip device as shown in FIG. 33A. FIG. 34B illustrates a cutawayassembled side view of a stretchable open top chip device as shown inFIG. 33A.

In one embodiment, the present invention contemplates a tall channelstretchable open top chip device 3500 comprising: i) a fluidic cover3510 comprising an open region 3504; ii) a top structure 3520 comprisingan upper microchannel 3534 attached to the fluidic cover 3510; iii) abottom structure 3525 comprising a lower microchannel 3536 attached tothe top structure 3520; and iv) a membrane 3540 layer between the bottomstructure 3525 and the top structure 3520. In one embodiment, the openregion 3504, upper microchannel 3534 and lower microchannel 3536 areconfigured to at least partially overlay each other. FIG. 35A and FIG.35B. Although not intended to be limiting, the tall channel stretchableopen top chip device 3500 may also comprise a vacuum port pair and/orinlet/outlet ports as shown and described above.

Although it is not necessary to understand the mechanism of an inventionit is believed that a tall channel strechable open top chip designrepresents a fundamental shift in architecture as compared toconventional “tissue-on-a-chip” designs. It is further believed thatthis tall channel open top design incorporates an openable lid fordirect access to the top channel that allows for the ability to loadthick gel matricies as well as micro-patterning of the gel.

Furthermore, the open top strechable test platforms as contemplatedherein are useful for biological interfaces, co-cultures, multiple celltype cultures, tissue streching, 3D scaffold models, micro-patterningand tissue chips including, but not limited to, skin, lung and intestine(e.g., gut). In one embodiment, a tall channel open top strechabledevice may have the following specifications:

Body Material PDMS Sylgard 184 Membrane Material PDMS Sylgard 184Dimensions Width 15.87 mm Length 35.87 mm Height 5.85 mm Top ChannelDimensions Top Channel Width 1000 µm Top Channel Height (closed) 1000 µmTop Channel Height (open) 2000 µm Top ChannelVolume 28.041 mm³ TopCulture Area 28.0 mm² Bottom Channel Dimensions Bottom Channel Width1000 µm Bottom Channel Height 200 µm Bottom Channel Volume 5.584 mm³Bottom Culture Area 24.5 mm² Membrane Dimensions Pore Diameter 7.0 µmPore Spacing 40 µm (hexagonally packed) Thickness 50 µm Co-culture Area17.1 mm² Minimum Imaging Distance (top of membrane) 850 mm

In one embodiment, the present invention contemplates a fully assembledstretchable open top microfluidic device 3600 comprising a fluidic cover3610 comprising microfluidic channel 3608, a first vacuum port 3630 anda second vacuum port 3632, wherein the microfluidic channel 3608terminates at either end an an inlet port 3614 and an outlet port 3616,respectively. FIG. 36 .

A first cross-sectional view across plane A of FIG. 36 presents an opentop microfluidic device 3700 in an assembled configuration comprising afluidic cover 3710 attached to a membrane 3740, wherein the membrane3740 overlays an open region 3704 (shown as hidden open region 3604 inFIG. 36 ) within a top structure 3720 that is attached to a bottomstructure 3725. FIG. 37A. A second cross-section view across plane A ofFIG. 36 presents an open top microfluidic device 3700 in a separatedconfiguration where a fluidic top 3710 comprising a membrane 3740 isremoved from top structure 3720 thereby providing access to an openregion 3704, wherein a microfluidic channel 3608 is configured withinthe fluidic cover 3710. FIG. 37B.

A third cross-sectional view across plane A of FIG. 36 presents an opentop microfluidic device 3800 in an assembled configuration comprising afluidic cover 3810 attached to a membrane 3840, wherein the membrane3840 overlays an open region 3804 (shown as hidden open region 3604 inFIG. 36 ) within a top structure 3820 that is attached to a bottomstructure 3825. FIG. 38A. A fourth cross-section view across plane A ofFIG. 36 presents an open top microfluidic device 3800 in a separatedconfiguration where a fluidic top 3810 comprising a membrane 3840 isremoved from top structure 3820 thereby providing access to an openregion 3804, wherein a microfluidic channel 3608 is configured totraverse between fluidic cover 3810 and top structure 3820. FIG. 38B.

EXPERIMENTAL Example 1 - Keratinocyte and Fibroblast Cell Culture

This example describes the preparation of keratinocytes, and inparticular human foreskin keratinocytes (HFKs). An aliquot of Lonza GoldKGM media (Lonza 192060) is placed in a 50ml tube (i.e. with 1 cryovialof HFK cells, one needs 12 ml for the flask, 10 ml for the washing stepand 1 to 5 ml to break the pellet for a total of about 25 ml). Themedium is warmed by putting it into the water bath for 5-10 min and thentransferred inside the sterile hood. The needed number of 15 and 50 mlconical tubes are prepared, along with the needed number of flasks.These are filled with the appropriate amount of Lonza medium.

To thaw the HFKs, a cryovial is removed from the liquid nitrogencontainer and transferred into the basket containing dry ice. Thecryovial is placed into the water bath until the freezing medium insideit is completely melted. The cryovial is sprayed with ethanol andbrought to the sterile hood. The cryovial is opened in the hood and thecontents are collected from the cryovial (freezing medium + cells) usinga 1000 µl pipette. The contents are transferred into the 15ml conicaltube containing Lonza Gold KGM medium previously warmed. This conicaltube is closed and then tilted to mix. Thereafter, it is centrifuged at1000 rpm for 5 minutes. The conical tube is sprayed with ethanol andreturned to the sterile hood. It is opened and the supernatant iswithdrawn, leaving the cell pellet. The pellet is re-suspended usingfresh pre-warmed Lonza Gold KGM and the mixture is transferred to aflask (or flasks), which were previously filled with Lonza Gold KGMmedium. The flasks are gently agitated to make sure that the mediumcovers the entire bottom surface. The flasks are then transferred to theincubator. The keratinocytes are fed with new media approximately everyother day (about every 36 hours).

To thaw the fibroblasts, a cryovial is removed from the liquid nitrogentank and transferred into the basket containing dry ice. The cryovial isplaced into the water bath until the freezing medium inside it iscompletely melted. The cryovial is sprayed with ethanol and brought tothe sterile hood. The cryovial is opened in the hood and the contentsare collected from the cryovial (freezing medium + cells) using a 1000µl pipette. Tee contents are transferred into the 15 ml conical tubecontaining Lonza FGM-2 medium previously warmed. This conical tube isclosed and then tilted to mix. Thereafter, it is centrifuged at 1200 rpmfor 5 minutes. The conical tube is sprayed with ethanol and returned tothe sterile hood. It is opened and the supernatant is withdrawn, leavingthe cell pellet. The pellet is re-suspended using fresh pre-warmed LonzaFGM-2 and the mixture is transferred to a flask (or flasks) which werepreviously filled with Lonza FGM-2 medium. The flasks are gentlyagitated to make sure that the medium covers the entire bottom surface.The flasks are then transferred to the incubator. The fibroblasts arefed with new media approximately every other day (about every 36 hours).

For detaching the HFKs by trypsinization, the protocol is as follows.First, an aliquot Lonza Gold KGM (Lonza 192060), Lonza reagentsubculture reagent CC-5034 and E-medium (or variants) 10% FBS medium isplaced in 15 ml and 50 ml tubes. It is convenient to us 4 mls of Lonzareagent subculture reagent CC-5034 per T75 flask and to add 8 mls of 10%FBS medium to the flask (which corresponds to 2 ml for each ml ofreagent Lonza reagent subculture reagent CC-5034). The media and enzymesare warmed by putting it into the water bath for 5-10 min. The flaskcontaining HFK (typically when the cells are between 50 and 70%confluence) is removed from the incubator, sterilized on the outsidewith ethanol, and transferred into the hood. The flask is opened and thethe Lonza Gold KGM medium is aspirated, being careful to not scratch thebottom flask surface where the cells are attached. Fresh pre-warmedLonza Gold KGM medium (e.g. 5 mls) is then added to wash the cells. Thismedia is also aspirated carefully. Then, 4 ml of 0.05% trypsin/EDTA(Corning 25-052 CL) is added to the flask and the flask is returned tothe incubator. The detaching cells can be monitored using the microscopeif desired. As a rule of thumb, keratinocytes should detach in about 2-3minutes. Longer exposure to Lonza subculture reagent CC-5034 (or 0.05EDTA trypsin Invitrogen 25200-056) could damage keratinocytesirreversibly. When the cells detach completely, the outside of the flaskis sterilized and brought to the hood. The flask is opened and 8 ml of10% FBS E-medium (or variants) is added to the flask (2ml for each ml of0.05 EDTA trypsin Corning 25-052-CL). Thereafter, the contents of theflask are conveniently transferred to a 15 ml conical tube. The tube isclosed and centrifuged at 1000 rpm for 5 min. The tube is thensterilized with ethanol, returned to the hood and opened. Thesupernatant is gently aspirated, being careful not to disturb the cellpellet. After the supernatant is removed, the pellet is re-suspendedusing fresh pre-warmed Lonza Gold KGM medium. The mixture is thentransferred to the flask/flasks, which were previously filled with LonzaGold KGM medium. The flasks are gently agitated to make sure that themedium covers the entire bottom surface, and they are returned to theincubator. Feeding is as stated above.

For detaching the fibroblasts by trypsinization, the protocol is asfollows. An aliquot of Lonza FGM-2 medium (Lonza CC-3132), Lonza reagentsubculture reagent CC-5034 and 10% FBS medium is added in 15 ml and 50ml tubes. It is convenient to use 4 ml Lonza reagent subculture reagentCC-5034 per T75 flask and 8 ml of 10% FBS medium to the flask (whichcorresponds 2 ml for each ml of reagent Lonza reagent subculture reagentCC-5034). The media and enzymes are warmed by putting them into thewater bath for 5-10 min. The flask containing fibroblasts (typicallywhen the cells are between 50 and 70% confluence) is removed from theincubator, sterilized on the outside with ethanol, and transferred intothe hood. The flask is opened and the media is aspirated gently, beingcareful to not scratch the bottom flask surface containing the celllayer. 5 ml of fresh PBS is added to wash the cells (this can be donetwice). The PBS is aspirated carefully, and 4 ml of 0.05% trypsin/EDTA(Lonza CC-5012) is added and the flask is returned to the incubator. Thedetaching cells can be monitored using the microscope if desired. As arule of thumb, fibroblasts should detach in about 2-3 minutes. Longerexposure could damage the cells irreversibly. When the cells detachcompletely, the outside of the flask is sterilized and brought to thehood. The flask is opened and 8 ml of Trypsin NeutralizingSolution(CC-5002) [2 ml for each ml of 0.05% trypsin/EDTA (LonzaCC-5002)] is added. The flask contents are transferred to a 15 mlconical tube and this tube is centrifuged at 1000 rpm for 5 min. Thetube is sterilized with ethanol and returned to the hood. Thesupernatant is aspirated, being careful not to disturb the cell pellet.Then, the pellet is re-suspended using fresh pre-warmed Lonza FGM-2medium and the contents are transferred to the flask/flasks, which werepreviously filled with Lonza FGM-2 medium. The flasks are gentlyagitated to make sure that the medium covers the entire bottom surfaceand then returned to the incubator. Feeding is as indicated above.

Example 2 - Embedding Cells in the Dermal Layer

For embedding fibroblasts into the dermal layer (e.g. gel matrix), theprotocol is as follows. First, the fibroblasts are detached using thetrypsinization protocol described above. However, the pellet isre-suspended in complete E-medium low calcium (0.6 mM Ca⁺⁺),supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 2%penicillin/streptomycin (invitrogen 15140-122) and then added back tothe flasks, where they are allowed to reach 50-60% confluence. Onceagain, the fibroblasts are detached according to the protocol describedabove. Once re-suspended, they are embedded into the dermal layer. FromDay 0 to Day 1-2, the cells in the dermal layer are fed using completeE-medium low calcium (0.6 mM Ca⁺⁺), supplemented with 0.5% (V/V) FBS(Invitrogen 16140071) and 100 µm ascorbic acid, RM/TI transglutaminase50 µg/ml. From Day 1-2 to Day 3-4, the cells in the dermal layer are fedusing complete E-medium low calcium (1.2 mM Ca⁺⁺), supplemented with0.5% (V/V) FBS (Invitrogen 16140071) and 100 µm ascorbic acid and RM/TItransglutaminase 50 µg/ml. From Day 14-18 on, the cells in the dermallayer are fed using complete cornification medium (1.8 mM Ca⁺⁺),supplemented with 5% (V/V) FBS (Invitrogen 16140071) and 100 µm ascorbicacid and RM/TI transglutaminase 50 µg/ml.

Example 3 - Preparing the Dermal Layer

First, pipette tips are cooled by putting into refrigerator for 15-30min (Pipettes need to be cold when working with rat-tail type I collagenin order to avoid coagulation). Both the pipette tips and the ECM matrixshould stay in the ice box during the procedure.

In order to calculate the final volume of rat-tail type I collagenmixture needed, one calculates the number of dermal equivalent culturesthat are needed. This calculation is based on 12 well + 3 extra (thoseare needed to compensate for the ECM matrix that adheres to the surfaceof pipette). Where 2×10⁴ neonatal or adult Human Foreskin Fibroblast perraft are employed and 12+3 rafts are prepared, one needs 15X 2×10⁴ =30×10⁴ fibroblasts (or 300,000 fibroblasts).To impede fibroblastsproliferation, one can irradiate the fibroblast with 70 Gy.

Now, to make 150 µl/raft X (12+3) rafts = 2.25 ml. 10% 10X DMEM orvariants *= 0.225 ml or 225 µl. 10% reconstruction buffer ⁺ = 0.225 mlor 225 µl. 80% ECM matrix = 1.8 ml or 1800 µl. (1.8 ml ECM matrix x 2.4× 10 1N NaOH (1 M))= 43.2 µl 1 M NaOH (1 M) (NaOH makes ECM matrix tocoagulate). This is put into INCUBATOR 37° C. for 2-4 Hours.

One can trypsinize the fibroblasts using 0.05% trypsin/EDTA (Corning25-052 CL) according to protocol described above. One can thenre-suspend the fibroblast pellet in the predetermined amount of 10X DMEMor variants. This is mixed with the necessary amount of reconstitutionbuffer. (Note: best results are obtained when fibroblasts are collectedin active growth phase, which occurs when fibroblast are between 50 and70% confluence).

100 µl ECM + fibroblast are added to each well and this is incubated(37° C. for 2 Hours). Thereafter, 100 µl of E medium is added to the topof each collagen gel. 100 µl of E medium + RM TG* is then added to thebottom of each collagen gel. This is incubated (37° C. for 12-16 Hours).

A variety of collagen containing matrices are contemplated for making anartificial derma and ECM to embed fibroblasts:

-   Tropoelastin : Collagen I: Collagen III: Dermatan sulfate (1 mg:3    mg:3 mg:0.5 mg)-   Col I (3 mg/ml) / Elastin (3 mg/ml)-   Col I (3 mg/ml) / Elastin (1 mg/ml)-   Col I (10 mg/ml) / MaxGEL-   Col I (3 mg/ml) / Elastin (3 mg/ml) 1: 1 MaxGel-   Col I (3 mg/ml) / Elastin (3 mg/ml) / Col III (3 mg/ml) 1: 1: 1-   MaxGel-   Col I (10 mg/ml) / Elastin (10 mg/ml)

Additional embodiments are contemplated:

1. A device comprising i) a chamber, said chamber comprising a lumen,said lumen positioned under ii) a removable top and above iii) a porousmembrane, said membrane positioned above one or more iv) fluidicchannels.

2. The device of Claim 1, further comprising a gel matrix.

3. The device of Claim 2, further comprising parenchymal cells on or inthe gel matrix, or both.

4. The device of Claim 3, wherein said parenchymal cells are selectedfrom the group consisting of epithelial cells of the lung and epithelialcells of the skin.

5. The device of Claim 4, wherein said epithelial cells of the lung areselected from the group consisting of alveolar epithelial cells andairway epithelial cells.

6. The device of Claim 4, wherein said epithelial cells of the skincomprise keratinocytes.

7. The device of Claim 1, further comprising positioned on the bottom ofthe membrane so as to be in contact with the fluidic channels.

8. The device of Claim 7, wherein the endothelial cells are primarycells.

9. The device of Claim 8, wherein said primary cells are small vesselhuman dermal microvascular endothelial cells.

10. The device of Claim 8, wherein said primary cells are humanumbilical vein endothelial cells.

11. The device of Claim 8, wherein said primary cells are bonemarrow-derived endothelial progenitor cells.

12. The device of Claim 6, wherein said keratinocytes are epidermalkeratinocytes.

13. The device of Claim 6, wherein said keratinocytes are human foreskinkeratinocytes.

14. The device of Claim 1, wherein said device is a microfluidic deviceand said fluidic channels are microfluidic channels.

15. A device comprising i) a chamber, said chamber comprising a lumen,said lumen comprising ii) a gel matrix, said gel matrix comprisingparenchymal cells, said gel matrix positioned above iii) a porousmembrane, said membrane comprising endothelial cells in contact with iv)fluidic channels.

16. The device of Claim 15, wherein said parenchymal cells are selectedfrom the group consisting of epithelial cells of the lung and epithelialcells of the skin.

17. The device of Claim 16, wherein said epithelial cells of the lungare selected from the group consisting of alveolar epithelial cells andairway epithelial cells.

18. The device of Claim 16, wherein said epithelial cells of the skincomprise keratinocytes.

19. The device of Claim 18, further comprising fibroblasts within thegel matrix, wherein the keratinocytes are on top of the gel matrix.

20. The device of Claim 19, wherein the keratinocytes comprise more thanone layer on top of the gel matrix.

21. The device of Claim 15, wherein the endothelial cells are primarycells.

22. The device of Claim 21, wherein said primary cells are small vesselhuman dermal microvascular endothelial cells.

23. The device of Claim 21, wherein said primary cells are humanumbilical vein endothelial cells.

24. The device of Claim 21, wherein said primary cells are bonemarrow-derived endothelial progenitor cells.

25. The device of Claim 18, wherein said keratinocytes are epidermalkeratinocytes.

26. The device of Claim 18, wherein said keratinocytes are humanforeskin keratinocytes.

27. The device of Claim 15, further comprising an open region in contactwith at least one of said gel, said membrane, said parenchymal cells orsaid endothelial cells.

28. A method of testing a drug, comprising 1) providing a) a candidatedrug and b) device comprising i) a chamber, said chamber comprising alumen, said lumen positioned above ii) a porous membrane, said membranecomprising parenchymal cells and positioned above one or more iii)fluidic channels; and 2) contacting said parenchymal cells with saidcandidate drug.

29. The method of Claim 28, wherein said parenchymal cells are selectedfrom the group consisting of epithelial cells of the lung and epithelialcells of the skin.

30. The method of Claim 29, wherein said epithelial cells of the lungare selected from the group consisting of alveolar epithelial cells andairway epithelial cells.

31. The method of Claim 29, wherein said epithelial cells of the skincomprise keratinocytes.

32. The method of Claim 31, further comprising fibroblasts within thegel matrix, wherein the keratinocytes are on top of the gel matrix.

33. The method of Claim 28, wherein said chamber lacks a covering andsaid candidate drug is introduced into said lumen under conditions suchthat said parenchymal cells are contacted.

34. The method of Claim 28, wherein said candidate drug is in anaerosol.

35. The method of Claim 28, wherein said candidate drug is in a paste.

36. The method of Claim 28, wherein said device further comprises aremovable top and said method further comprises, prior to step 2),removing said removable top.

37. A method of testing an agent comprising 1) providing a) an agent andb) microfluidic device comprising i) a chamber, said chamber comprisinga lumen, said lumen comprising ii) a gel matrix comprising cells in, onor under said gel matrix, said gel matrix positioned above iii) a porousmembrane and under iv) a removable cover, said membrane positioned aboveone or more v) fluidic channels; 2) removing said removable cover; and3) contacting said cells in, on or under said gel matrix with saidagent.

38. The method of Claim 37, wherein said agent is in an aerosol.

39. The method of Claim 37, wherein said agent is in a paste.

40. The method of Claim 37, wherein said agent is in a liquid, gas, gel,semi-solid, solid, or particulate form.

41. A device comprising i) a chamber, said chamber comprising a lumenand projections into the lumen, said lumen comprising ii) a gel matrixanchored by said projections, said gel matrix positioned above iii) aporous membrane, said membrane positioned above one or more iv) fluidicchannels.

42. The device of Claim 41, wherein fibroblasts are within the gelmatrix and keratinocytes are on top of the gel matrix.

43. The device of Claim 42, wherein the keratinocytes comprise more thanone layer on top of the gel matrix.

44. The device of Claim 41, wherein a layer of endothelial cells ispositioned on the bottom of the membrane so as to be in contact with thefluidic channels.

45. The device of Claim 44, wherein the endothelial cells are primarycells.

46. The device of Claim 45, wherein said primary cells are small vesselhuman dermal microvascular endothelial cells.

47. The device of Claim 45, wherein said primary cells are humanumbilical vein endothelial cells.

48. The device of Claim 45, wherein said primary cells are bonemarrow-derived endothelial progenitor cells.

49. The device of Claim 42, wherein said keratinocytes are epidermalkeratinocytes.

50. The device of Claim 42, wherein said keratinocytes are humanforeskin keratinocytes.

51. The device of Claim 41, further comprising a removable cover.

52. The device of Claim 41, wherein said device is a microfluidic deviceand said fluidic channels are microfluidic channels.

53. A microfluidic device comprising i) a chamber, said chambercomprising a lumen and projections into the lumen, said lumen comprisingii) a gel matrix anchored by said projections, said gel matrixcomprising fibroblasts and keratinocytes, said gel matrix positionedabove iii) a porous membrane, said membrane comprising endothelial cellsin contact with iv) microfluidic channels.

54. The device of Claim 53, wherein the membrane is above said fluidicchannels and wherein the layer of endothelial cells is positioned on thebottom of the membrane so as to be in contact with the fluidic channels.

55. The device of Claim 53, wherein the fibroblasts are within the gelmatrix and the keratinocytes are on top of the gel matrix.

56. The device of Claim 55, wherein the keratinocytes comprise more thanone layer on top of the gel matrix.

57. The device of Claim 53, wherein the endothelial cells are primarycells.

58. The device of Claim 57, wherein said primary cells are small vesselhuman dermal microvascular endothelial cells.

59. The device of Claim 57, wherein said primary cells are humanumbilical vein endothelial cells.

60. The device of Claim 57, wherein said primary cells are bonemarrow-derived endothelial progenitor cells.

61. The device of Claim 53, wherein said keratinocytes are epidermalkeratinocytes.

62. The device of Claim 53, wherein said keratinocytes are humanforeskin keratinocytes.

63. The device of Claim 53, wherein said matrix comprises collagen.

64. The device of Claim 53, wherein said collagen matrix is between 0.2and 6 mm in thickness.

65. A method of testing a drug on keratinocytes, comprising 1) providinga) a candidate drug and b) microfluidic device comprising i) a chamber,said chamber comprising a lumen and projections into the lumen, saidlumen comprising ii) a gel matrix anchored by said projections, said gelmatrix comprising fibroblasts and keratinocytes, said gel matrixpositioned above iii) a porous membrane, said membrane comprisingendothelial cells in contact with iv) fluidic channels; and 2)contacting said keratinocytes with said candidate drug.

66. The method of Claim 28, wherein the fibroblasts are within the gelmatrix and the keratinocytes are on top of the gel matrix.

67. The method of Claim 28, wherein said chamber lacks a covering andsaid candidate drug is introduced into said lumen under conditions suchthat said keratinocytes are contacted.

68. The method of Claim 28, wherein said candidate drug is in anaerosol.

69. The method of Claim 28, wherein said candidate drug is in a paste.

70. The method of Claim 28, wherein said microfluidic device furthercomprises a removable top and said method further comprises, prior tostep 2), removing said removable top.

71. The method of Claim 28, wherein said microfluidic device furthercomprises an open region in contact with at least one of said gelmatrix, said membrane, said keratinocytes or said endothelial cells.

72. A method of testing an agent comprising 1) providing a) an agent andb) microfluidic device comprising i) a chamber, said chamber comprisinga lumen and projections into the lumen, said lumen comprising ii) a gelmatrix anchored by said projections and comprising cells in, on or undersaid gel matrix, said gel matrix positioned above iii) a porous membraneand under iv) a removable cover, said membrane positioned above one ormore v) fluidic channels; 2) removing said removable cover; and 3)contacting said cells in, on or under said gel matrix with said agent.

73. The method of Claim 72, wherein said agent is in an aerosol.

74. The method of Claim 72, wherein said agent is in a paste.

75. The method of Claim 72, wherein said agent is in a liquid, gas, gel,semi-solid, solid, or particulate form.

Still additional embodiments are contemplated:

28. A fluidic cover comprising a fluidic channel, said fluidic coverconfigured to engage a microfluidic device.

29. The fluidic cover of Claim 28, wherein said microfluidic devicecomprises an open chamber, and wherein said fluidic cover configured tocover and close said open chamber.

30. The fluidic cover of Claim 28, further comprising one or moreelectrodes.

31. An assembly comprising a fluidic cover comprising a fluidic channel,said fluidic cover detachably engaged with a microfluidic device.

32. The assembly of Claim 31, wherein said microfluidic device comprisesan open chamber, and wherein said fluidic cover configured to cover andclose said open chamber.

33. The assembly of Claim 32, wherein said open chamber comprises anon-linear lumen.

34. The assembly of Claim 33, wherein said non-linear lumen is circular.

35. The assembly of Claim 31, wherein said fluidic cover furthercomprises one or more electrodes.

36. A method of making an assembly, comprising: a) providing a fluidiccover comprising a fluidic channel, said fluidic cover configured toengage b) a microfluidic device, said microfluidic device comprises anopen chamber, and wherein said fluidic cover configured to cover andclose said open chamber; and b) detachably engaging said microfluidicdevice with said fluidic cover so as to make an assembly.

37. The method of making an assembly of Claim 36, wherein said openchamber comprises a non-linear lumen.

38. The method of making an assembly of Claim 37, wherein saidnon-linear lumen is circular.

39. The method of making an assembly of Claim 36, wherein said fluidiccover further comprises one or more electrodes.

1-30. (canceled)
 31. A layered structure, comprising: a) fluidicchannels covered by b) a porous membrane, said membrane positioned belowc) a gel matrix, said gel matrix comprising d) cells in or under the gelmatrix, and e) a removable fluidic cover over the gel matrix, saidfluidic cover comprising a fluidic channel configured to perfuse saidcells.
 32. The layered structure according to claim 31 in the form of adevice comprising a lumen, said lumen positioned under the e) removablecover and above the a) fluidic channels, b) porous membrane, c) gelmatrix and d) cells.
 33. The layered structure of claim 32, wherein saidcells are parenchymal cells on or in the gel matrix, or both.
 34. Thelayered structure of claim 33, wherein said parenchymal cells areselected from the group consisting of epithelial cells of the lung andepithelial cells of the skin.
 35. The layered structure of claim 34,wherein said epithelial cells of the lung are selected from the groupconsisting of alveolar epithelial cells and airway epithelial cells. 36.The layered structure of claim 34, wherein said epithelial cells of theskin comprise keratinocytes.
 37. The layered structure of claim 31,further comprising endothelial cells positioned on the bottom of themembrane so as to be in contact with the fluidic channels.
 38. Thelayered structure of claim 37, wherein the endothelial cells are primarycells.
 39. The layered structure of claim 38, wherein said primary cellsare small vessel human dermal microvascular endothelial cells.
 40. Thelayered structure of claim 38, wherein said primary cells are humanumbilical vein endothelial cells.
 41. A method of making an assembly,comprising a) providing a fluidic cover comprising a fluidic channel,said fluidic cover configured to engage a microfluidic device, saidmicrofluidic device comprising an open chamber, and wherein said fluidiccover is configured to cover and close said open chamber; and b)detachably engaging said microfluidic device with said fluidic cover soas to make an assembly.
 42. The method of making an assembly of claim41, wherein said open chamber comprises a non-linear lumen.
 43. Themethod of making an assembly of claim 42, wherein said non-linear lumenis circular.
 44. The method of making an assembly of claim 41, whereinsaid fluidic cover further comprises one or more electrodes.
 45. Themethod of claim 41, further comprising introducing fluid into saidfluidic channel of said fluidic cover, whereby fluid perfuses said openchamber.